Picking through centuries-old rubbish, masonry and discarded body parts beneath an abandoned Tuscan church, an Italian historian believes she has solved one of history's great crime mysteries.

For more than four centuries, researchers have puzzled over the fact that Francesco I Medici, the son of the first Grand Duke, Cosimo, died within hours of his wife in October 1587. Legend had it they were poisoned by his brother and successor, a cardinal.

Modern historians have tended to settle for the more down-to-earth explanation that they died of malaria. But Donatella Lippi, an associate professor at the University of Florence, told the Guardian yesterday that she and other researchers had established beyond doubt that Francesco was poisoned and that evidence from "mountains of debris" underneath the deconsecrated church strongly suggested his wife was too.

Prof Lippi said that when she came across the remains that were to yield the vital clues "I very nearly had a heart attack".

The details of the investigation have been published in the BMJ.

Prof Lippi is historical adviser to a project in which the bodies of numerous members of the Medici dynasty, including Francesco, have been dug up in search of new evidence on their lives and deaths.

She said a document she found while researching for the project had indicated that a postmortem had been carried out on the embalmed bodies of the Grand Duke and his wife, a Florentine noblewoman, Bianca Cappello.

The document, from the diocesan archives of the city of Pistoia, showed that the organs extracted during the autopsy had been put into terracotta jars and placed under the church of Santa Maria a Buonistallo, near the villa where they had died.

The church, though no longer used for worship, still belonged to the ecclesiastical authorities and Prof Lippi was able to get permission to go into the basement with a team of building workers.

"What we found was not a room but a kind of tunnel full of debris. I decided to go through it with my bare hands so I could actually feel the difference between the stones and dust and any organic matter we might find. It was a terrible job," Prof Lippi said.

Her search yielded part of a human liver "the size of a hazelnut" and two other body parts that have defied identification. Tests showed the liver was that of a man and its DNA matched that taken from remains in Francesco's tomb. The other body parts belonged to a woman and, like the fragment of liver, they revealed high concentrations of arsenic.

Francesco's brother, Cardinal Ferdinando, had been in danger of being excluded from the succession. In his letters to the papal court, he put the Grand Duke's illness down to his eating habits and said Bianca was sick with grief because of her husband's condition.