Given the global threat of antimicrobial resistance, it is imperative that veterinary graduates are effective antimicrobial stewards. Veterinary students learn the principles of antimicrobial stewardship explicitly, through pre-clinical coursework, and implicitly, through the cases they each encounter on clinical rotations. We aimed to understand the influence of pre-clinical versus clinical learning on veterinary students’ knowledge and awareness of antimicrobial concepts to guide efforts to improve instruction in these areas. To assess knowledge acquisition and to explore student perceptions of antimicrobial stewardship, a standardized online survey was administered to Cornell University veterinary students at two timepoints: in August 2020 before clinical rotations ( N = 26 complete responses and N = 24 partial responses) and again in May 2021 after their clinical rotations ( N = 17 complete responses and N = 6 partial responses). Overall and section-specific confidence and knowledge scores were calculated, using pairwise deletion for incomplete responses. Students generally had low confidence in antimicrobial topics and correctly answered only half of knowledge questions correctly; they performed the best on antimicrobial resistance knowledge questions. There were no significant differences in knowledge or confidence after clinical rotations. On average, students had only read one antimicrobial stewardship guideline. Students reported that human health care providers contributed more to antimicrobial resistance than veterinarians. In conclusion, graduating veterinary students at our institution have significant knowledge gaps in critical principles that are essential to become antimicrobial stewards. Explicit instruction in antimicrobial stewardship is necessary in the pre-clinical and clinical coursework, and the practical use of antimicrobial stewardship guidelines should be emphasized.