This cross-sectional descriptive study aims to explore the knowledge, attitudes, and practices among pharmacy, dentistry, medicine, and nursing interns in Saudi Arabia regarding antibiotic use and antibiotic resistance. Interns received a soft copy of a structured and validated self-administered questionnaire using an online survey platform. A total of 266 interns responded to the questionnaire. On average, the participants achieved good scores in the knowledge and practice domains, followed by the attitude domain. The average knowledge percentage was 76.1% (SD 17.1) compared to 84.6% (SD 20.5) for practices and 61.5% (SD 23.2) for attitudes. The results suggest that pharmacy interns had insignificantly better overall scores compared to non-pharmacy interns for knowledge, attitudes, and practices. This study shows that the scores for knowledge, attitudes, and practices of pharmacy, dentistry, medicine, and nursing interns regarding antibiotic use and resistance were high overall. However, interns’ belief in their responsibility in preventing antibiotic resistance is lacking.