A better understanding of veterinary students’ perceptions, attitudes, and knowledge about antimicrobial stewardship and biosecurity could facilitate more effective education of future veterinarians about these important issues. A multicenter cross-sectional study was performed by administering a questionnaire to veterinary students expected to graduate in 2017 or 2018 in all Australian veterinary schools. Four hundred and seventy-six of 1246 students (38%) completed the survey. Many students were unaware of the high importance of some veterinary drugs to human medicine, specifically enrofloxacin and cefovecin (59% and 47% of responses, respectively). Fewer than 10% of students would use appropriate personal protective equipment in scenarios suggestive of Q fever or psittacosis. Students expected to graduate in 2018 were more likely to select culture and susceptibility testing in companion animal cases (OR 1.89, 95% CI 1.33–2.69, p < 0.001), and were more likely to appropriately avoid antimicrobials in large animal cases (OR 1.75, 95% CI 1.26–2.44, p = 0.001) than those expected to graduate in 2017. However, 2018 graduates were less likely to correctly identify the importance rating of veterinary antimicrobials for human health (OR 0.48, 95% CI 0.34–0.67, p < 0.001) than 2017 graduates. Students reported having a good knowledge of antimicrobial resistance, and combating resistance, but only 34% thought pharmacology teaching was adequate and only 20% said that teaching in lectures matched clinical teaching. Efforts need to be made to harmonize preclinical and clinical teaching, and greater emphasis is needed on appropriate biosecurity and antimicrobial stewardship.