During the Renaissance period, there was no unified Italy, but it was divided into a number of warring city-states, kingdoms and duchies (see above map). These places were ruled by powerful families like the Medici and Orsini who used both money and matrimonial alliances to their advantage. The Pope, during that time, was Pope Alexander VI (Rodrigo Borgia) and one of his sons, Cesare Borgia, was governing the region of Romagna.  During that period, Leonardo da Vinci worked for Cesare Borgia as his military engineer. Also present in the same place was Niccolò Machiavelli, who was the Florentine secretary.
The Borgias were one of the most notorious families in European history and there is no shameful act that has not happened under their watch. When Rodrigo was around 25, his maternal uncle Pope Callixtus III, made him a bishop. The word nepotism (nepotis – Latin for  nephew) comes from this system which was common during that period. Rodrigo became Pope in the year Christopher Columbus set off to on his voyage to paradise.
During this period, alum, which was an important dye for the cloth industry, was discovered in the Roman hills. Previously it had to be imported from Turkey and following the fall of Constantinople in 1452, it was no longer an option. The revenue brought by the sale of alum was augmented by the sale of indulgences, a practice against which Martin Luther would revolt. This wealth was quite useful for Rodrigo to secure the future of his illegitimate children. He also ruled by terror; it was not considered a good idea to come in the path of the Borgias as murders and poisoning were common.
Among his illegitimate children, Rodrigo, who fathered when he was the Pope and also kept a mistress, had great ambitions for Cesare who was given command of the papal army. The novel is set in motion with the murder of his elder brother Juan and Cesare along with several Roman barons are suspect. This was followed by a series of grotesque murders of women who are considered as prostitutes and witches and their body parts are dispersed in carefully arranged patterns along the countryside.
To find the killer, the Pope sends Madonna Damiata, a courtesan who was the lover of the murdered Juan. At Imola, she meets Machiavelli and da Vinci, who work with her in solving the mystery. The first one-third of the book is written as a first person account by Damiata as she meets the various players in Cesare’s court and gets familiar with the vastly different techniques of the scientist and the man who studies people.
While The Secret Supper focused on Leonardo as an artist, this book is about the scientific methods he uses. In Imola, he is all about dissecting corpses, quantifying everything, creating maps and finding patterns based on that data and not guesswork. One of the fascinating parts of the book is when he disagrees with  Machiavelli on his techniques; his technique is based on analyzing historical events and the men who shaped them. Even though the times have changed, the nature of men remain the same, he argues and Leonardo cannot agree to that since it is of subjective nature.
During a ceremony at the place of a Romagnole witch, Damiata disappears and the remaining two-thirds of the book is a first person narrative by  Machiavelli. There is urgent need to find the killer because Cesare is about to make a pact with the Roman barons and it is possible that they might advance to  Machiavelli’s homeland of Florence.
All the characters in the book are real historical characters and all of them did what the historical record tells us. The missing part is why they did those things and Ennis fills those gaps. In an essay, Michael Ennis wrote

This evidence brought my sleuthing-geniuses premise squarely back into the domain of documented history: I had discovered a true crime story – involving, as it turns out, a brilliant serial killer–interlaced with one of history’s pivotal political events. Although this was a story Machiavelli, for very good reasons, decided to keep to himself, The Prince contains artifacts of it, once you know what you are looking for. As Machiavelli confesses to us at the beginning of his narrative, there is a “terrifying secret I deliberately buried between the lines of The Prince.” The words are my creation, but they are based on admissions that Machiavelli made later in his life. The truth that can be found between the lines of The Prince – a revelation of man’s capacity for evil far more ghastly than anything Machiavelli wrote explicitly in the text–is no mere fictional invention. With consequences that have resounded throughout the subsequent course of Western culture and history, the dreadful secret of The Prince is all too real.

It is also a great character study of Machiavelli  and shows how he came up with the concepts he later wrote in Prince. Even though the book looks a bit disconnected at some points, it is a great read and gives you a good account of the Borgias and politics of 16th century Italy. Also thanks to this book, I came across two television series (1, 2) based on this period.
References

1. BBC. In Our Time. The Borgias