Antimicrobial resistance (AMR) is a global threat to human health. Education on antibiotics is essential for AMR prevention, and training should be provided for undergraduate pharmacy students. This study evaluated the knowledge regarding antibiotic use and AMR among fourth-year Japanese pharmacy students and the effect of a lecture on treating infectious diseases with antibiotics had on their knowledge. A questionnaire survey was conducted, and the responses were recorded before and after participants attended the lecture. A small subset of the prelecture survey questions was used for the postlecture survey. From a total of 540 participants, 330 and 234 responses were collected before and after the lecture, respectively. In the prelecture survey, 39.4% of the participants incorrectly answered that antibiotics can effectively treat the common cold, 13.3% had taken leftover antibiotics, and 17.3% had taken antibiotics prescribed for their family members or others. Furthermore, the prelecture survey data showed that the mean number (± standard deviation) of correct answers across the eight questions on treatment and diagnosis of infectious diseases and antibiotics was 2.21 ± 1.64. However, in the postsurvey, this figure increased to 5.00 ± 1.82. Although the lecture improved their knowledge to some extent, the results suggested that fourth-year pharmacy students have inaccurate knowledge regarding the appropriate use of antibiotics and AMR. Therefore, it is necessary to improve early-year undergraduate pharmacy education on antibiotics in Japan.