Imane Fadil, 33, died in a Milan hospital on 1 March, a month after being admitted with stomach pains.
At the time she told friends and her lawyer that she had been poisoned.
She testified at the 2012 trial of Mr Berlusconi, who was accused of paying for sex with an underage prostitute.
- The many trials of Berlusconi
Mr Berlusconi was initially convicted but then acquitted on appeal. He was later convicted of tax fraud and sentenced to carry out community service.
Milan chief prosecutor Francesco Greco told Reuters news agency there were “several anomalies” in Ms Fadil’s medical records.
“The doctors have not identified with any certainty any pathology which can explain the death,” he said.
Mr Berlusconi’s reputation was tarnished by allegations about the parties at his private villa.
In October 2010, it emerged that Mr Berlusconi had called a police station asking for the release of a 17-year-old girl, Karima “Ruby” El Mahroug.
She was being held for theft and was also said to have attended his so-called “bunga-bunga” parties.
- An explanation of bunga bunga
In June 2013 he was found guilty of paying her for sex, and of abuse of power. The case was eventually overturned in 2014.
Mr Berlusconi has always maintained he is “no saint” but firmly rejects claims he has ever paid for sex with a woman, saying: “I never understood where the satisfaction is when you’re missing the pleasure of conquest.”
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