BACKGROUND: Dental students in Saudi Arabia are authorized to write prescriptions for antibiotics during practical training. Adverse side effects and resistance could result from inappropriate prescription. Accordingly, there is a need to evaluate the knowledge of dental students regarding guidelines and applications of antibiotic prescription. OBJECTIVES: To assess the knowledge and attitude toward guidelines and applications of antibiotic prescription among dental students and interns at multiple universities in Saudi Arabia. METHODS: A cross-sectional study was conducted among dental students in their final clinical years (4th to 6th year) and dental interns. The study data were collected using a valid and reliable structured questionnaire comprising three domains: 1) demographic characteristics, 2) knowledge, and 3) attitude toward antibiotic prescription for dental and systemic conditions. The data were analyzed and presented as frequency percentages, and the chi-square test was used to compare the knowledge and attitude items between the dental students and interns. The statistical significance level was set at p ≤ 0.05. RESULTS: A total of 248 participants (women: 55.6%, men: 44.4%) were included in the study. Approximately 21.8% were 4th year students; 17.7%, 5th year students; 12.9%, 6th year students; and 47.6%, interns. For most items, the knowledge level was relatively high, and the attitude was generally positive among the participants. Approximately, 87.1% had good knowledge about current guidelines for antibiotic prophylaxis, 83.9% about antibiotic prescription, and 95.2% about antibiotic resistance. The interns showed significantly higher knowledge levels and favorable attitude, particularly for guidelines and applications of antibiotic prescription and correct use of antibiotics for oral cases, than did the students. Amoxicillin was the most frequently prescribed antibiotic among the participants. CONCLUSION: The interns and 6th-year students demonstrated a relatively high knowledge level and positive attitude toward appropriate antibiotic prescriptions. However, deficiencies were observed among the students in their early clinical years, particularly for systemic conditions. These findings highlight the importance of implementing educational campaigns and providing guidelines to promote the appropriate use of antibiotics among dental students in their final clinical years.