In our series, including 1053 cases of infective endocarditis (IE) collected from 86 Infectious Diseases Centres in Italy between 1984 and 1999, we identified 34 cases (3.2%) with a polymicrobial etiology. Intravenous drug abuse was the most important risk factor for the development of polymicrobial IE. Twenty three patients had a left sided-IE and 6 patients had a right-sided IE. The most commonly encountered microorganisms were Staphylococci and Streptococci and the most frequently observed associations of microorganisms were those between Staphylococci and Gram-negative bacteria and between Staphylococci and fungi. Twelve patients (35.3%) underwent surgery, and 5 patients (16.7%) died. Polymicrobial endocarditis did not differ clinically from IE caused by a single microorganism, and the prognosis seems to be related to the site of infection and to some specific pathogens.