Background Antibiotics are widely used in dental practice, especially for endodontic infections. The present survey aimed to investigate the knowledge and practices of dental interns in Saudi Arabia regarding antibiotic prescription for endodontic treatment. Methods The present online questionnaire-based, cross-sectional study involved dental interns in private and public dental schools, Saudi Arabia. A pre-validated questionnaire was distributed to 900 dental interns via different social media platforms. The questionnaire consisted of 16 close-ended questions related to participants’ demographic data and knowledge and practices of antibiotic prescription in context of endodontic treatments. Data were managed and analyzed using IBM-SPSS version 25, and Chi-square test was used to compare between the groups. Results A total of 555 dental interns completed the questionnaire, giving a response rate of 61.1%. Overall, the surveyed participants revealed inadequate knowledge and unnecessary use of antibiotics during endodontic procedures. While majority of the participants (75.3%) correctly identified the first choice of antibiotics during endodontic treatments, a considerable proportion of the participants did not recognize the clinical indications of antibiotics in endodontic patients. Additionally, around one-fifth (18.9%) of the participants were unaware of the potential side effects of the prescribed antibiotics. Conclusion The present survey revealed unsatisfactory knowledge and practices of antibiotic prescription in context of endodontic therapy among Saudi dental interns. Therefore, dental schools in Saudi Arabia should address such a gap through updating the curriculum and integrating real-world clinical scenarios using problem-based learning. Additionally, periodic continuous education courses aiming at improving dental professionals’ knowledge about antibiotics and their clinical uses for endodontic therapy are highly encouraged.