Lazio had appealed the FIGC’s seven-month suspension of Lotito and a 150,000-euro fine imposed on the club on March 26.
Club doctors Ivo Pulcini and Fabio Rodia’s original 12-month bans were both confirmed by the FIGC court of appeal.
The penalties follow an FIGC investigation of positive tests at the Roman club in October and November.
Lazio were accused of failing to alert local health authorities when eight staff tested positive late October, on the eve of a Champions League match in Bruges, and then in November, before another Champions League match in St Petersburg.
UEFA requires players and staff to undergo tests before European matches.
At least one Lazio player was positive in UEFA tests but negative in Serie A tests in the same period.
The federation had also criticised Lazio for “not having prevented” three players from taking part in a squad training session on November 3, despite a positive test the day before.
Lazio are also said to have fielded players in Serie A who should have been in “a period of compulsory isolation, in the absence of symptoms, of at least 10 days”.