The 31-year-old football star was found dead in his hotel room in the northeastern Italian city of Udine on March 4, 2018, ahead of a Serie A match against Udinese.
Initial autopsy results declared the cause of death to be from bradyarrhythmia, a condition in which the heart slows to the point that it stops.
The doctors under investigation signed the fitness certificates which gave Astori the all-clear to play.
The two medics reportedly work for public bodies in Florence and Cagliari and are responsible for deeming athletes fit to play.
The investigation comes after Florentine daily La Nazione published details of two electrocardiogram tests carried out on Astori in July 2016 and July 2017
Both reportedly showed evidence that the player suffered from a ventricular extrasystole, or an irregular heartbeat.
Father to a two-year-old girl, Astori played 14 times for Italy during his career.
