Inappropriate use of antibiotics (AB) is a significant public health problem. Understanding the patterns of AB-taking behaviour helps in designing educational campaigns to curb the overuse of AB. The objective of this study was to determine the levels of knowledge, attitudes and practices (KAP) of AB usage among students and staff at the National Defence University of Malaysia. This cross-sectional study was conducted in July 2018. A self-administered questionnaire was used. Data were described by frequencies and percentages. Chi-square test was used to compare KAP levels between students and staff. The level of significance was established as p ≤ 0.05. This study revealed that fair level of knowledge about AB usage did not translate into positive attitudes and high levels of practices. Overall, students demonstrated poorer KAP regarding indiscriminate usage of AB than staff. Specifically, students had poorer knowledge about its role in ineffectiveness of treatment (p=0.013) and additional burden of medical cost to the patient (p=0.001). Additionally, students were more likely than staff to give the leftover AB to their friends if they become sick (p=0.024). The findings of this study may serve as a preliminary insight on the development of an effective intervention to improve attitudes and practices regarding AB usage.