We aimed to assess antibiotic usage and knowledge regarding antibiotics and antimicrobial resistance (AMR) among undergraduate students of the Universiti Brunei Darussalam (UBD), public university located in Brunei Darussalam. A cross-sectional study was performed using a self-administered questionnaire. The questionnaire was adapted from the World Health Organization’s (WHO) “Antibiotic resistance: Multi-country public awareness” survey distributed online. Students at the UBD were invited to participate in the online survey through internal email. The questionnaire consisted of five sections: demographic information, antibiotic usage, knowledge on antibiotics, antibiotic resistance (AMR), and use of antibiotics in agriculture. The data were analyzed descriptively and appropriate inferential statistics were used accordingly. A total of 130 students returned a completed questionnaire. The result of the study found that 51% (n = 66) of the students had good level of knowledge of antibiotic and antimicrobial resistance with a mean total knowledge score of nine out of 14. Of note, 76% (n = 99) of the respondents mistakenly believed that antibiotic resistance is the result of the body becoming resistant to antibiotics. Only 14% (n = 18) of the respondents were found to have poor knowledge on antibiotics and antimicrobial resistance in the study. Misconceptions in regards to the use of antibiotics for conditions related to viral illnesses like cold and flu (41%, n = 53) were noticed among the respondents in our study. Thus, improving knowledge on antibiotics is crucial to address these beliefs.