Background Antibiotic-resistant bacteria (ARB) have been recognized as one of the global health issues affecting humans, animals, and the environment. A lack of knowledge, negative attitudes, and irrational drug use can make significant contributions to the spread of ARB. This study aimed to assess the knowledge, attitudes, and practices (KAP) regarding antibiotic use and resistance among health science (HS) and non-health science (NHS) students and to determine the factors that influence their KAP concerning antibiotic use and resistance. Methods A cross-sectional study was conducted among 404 HS and NHS students in Southern Thailand from December 2021 to March 2022. The students who fulfilled the study inclusion criteria responded to a questionnaire that had five dimensions. Descriptive statistics were used to analyze the qualitative variables, and Fisher’s exact test was applied to compare the demographic variables between the HS and NHS students. Chi-squared tests were used to compare the percentages of each KAP question between the HS and NHS students. The KAP regarding antibiotic use and resistance for each variable were compared using the Mann–Whitney U test and Kruskal–Wallis H test. Spearman’s correlation test was used to estimate the correlation between the variables and KAP. Results A total of 404 (HS,162; NHS,242) students completed the self-administered questionnaire. The students’ highest score was for attitude, followed by practice and knowledge. Our findings revealed that the HS students had higher levels of KAP correlated with antibiotic use and resistance than the NHS students (P < 0.001). The higher KAP scores were among the more senior students, which indicates that instruction on antibiotics was effective in their curriculum. Conclusions Antibiotic use and resistance knowledge and attitudes should be conveyed to all university students via courses, seminars, or workshops. Such interventions could set the standard for rational antibiotic use as well as long-term prevention and control of antibiotic-resistant bacteria.