Infective endocarditis (IE) is an infection of the endothelial surface of the heart and heart valves with serious, even fatal, complications and that often requires long-term and expensive treatment. Dental procedures may lead to IE in high-risk patients. The aim of this study was to evaluate and compare the knowledge of general dentists and dental students concerning the prevention of IE in Hamadan, Iran. In this cross-sectional study, the awareness of general dentists and dentistry students concerning the prevention of IE was evaluated during 2010. A questionnaire was prepared and administered to 58 final-year dental students and 96 general dental practitioners in Hamadan. A total of 154 persons completed the questionnaire. The questionnaire consisted of some demographic questions and questions about awareness of IE in three sections. The gathered data were analyzed by using descriptive statistics, Pearson's chi-square test, Mann-Whitney test, and independent t-tests. The gathered data showed that dentistry students answered the questions about awareness of the prevention of IE more correctly than did general dentists. The overall knowledge of endocarditis prophylaxis among students and dentists was about 65% and 56%, respectively. The students' knowledge was better because 94.9% of the students had desired (acceptable) and relatively desired knowledge; this result for dentists, however, was 82.3%. In our study, the overall awareness level of the study population was moderate. Dentist and students believed that patients with prosthetic valves and previous IE were the most common cardiac disease cases that required prophylaxis. The most common prophylactic regimen was in accordance with the guidelines of the American Heart Association and was a single dose of 2 g amoxicillin 1 hour before treatment. The results indicated that gender had no effect on the level of knowledge; however, there was a statistically significant relationship between age and level of knowledge.