Background Infective endocarditis is a disease endangering the life of humans. Therefore, several prophylactic measures are needed to improve the protection of endocarditis-prone patients from bacteremia resulting from various dental actions. These measures range from developing the dental hygiene of endocarditis-prone individuals to trials of different antimicrobial agents. The objective of the study was to examine knowledge and practices of dentists in the Aseer Region, Saudi Arabia, regarding antibiotic prophylaxis against Infective endocarditis.Methods A cross-sectional study design in Aseer Region. Interviews were carried out, and 182 individuals participated in the data collection process. The 182 participants were interviewed at their workplaces. The data collection sheet was constructed based on the guidelines of the American Heart Association /American College of Cardiology (AHA/ACC, 2017).Results Dentists’ mean knowledge score was 17.5±3.7 (out of 24). The least correct responses regarding dental procedures that require prophylactic antibiotics were “root canal treatment” (30.8%). Regarding cardiac conditions that require prophylactic antibiotics, dentists’ least correct responses were “heart failure” (50%). Dentists’ mean knowledge scores differed significantly according to their age groups (p=0.032), nationality (p=0.002), education/qualification (p=0.002). Mean knowledge scores differed significantly according to dentists’ years of operation (p=0.018) and sources of information (p<0.001). Amoxicillin was the most regularly recommended antibiotic (90.7%), while 86.8% correctly stated 30-60 minutes before the procedure as the time for prophylactic antibiotic administration.Conclusions Knowledge of dentists in the Aseer Region regarding the use of preventive drugs for the control and prevention of I.E. is suboptimal. The inclusion of latest AHA guidelines into the dentistry curricula is highly recommended.