Introduction Antimicrobial resistance (AMR) is a global public health problem that has led to increased morbidity and mortality, especially in low- and middle-income countries such as Zambia. This study evaluated AMR knowledge, attitudes and practices among pharmacy personnel and nurses at Ndola Teaching Hospital, Zambia’s second-largest hospital. Methods A descriptive cross-sectional study was conducted among 263 participants using a structured questionnaire. Data analysis was performed with IBM SPSS version 23.0. All statistical tests were conducted at a 95% confidence level. Univariate analysis was used to determine differences in knowledge, attitudes and practices on AMR between pharmacy personnel and nurses. Results Of the 263 participants, 225 (85.6%) were nurses and 38 (14.4%) were pharmacy personnel. Compared with nurses, pharmacy personnel had better knowledge of the spread of resistant bacteria from one person to another (P = 0.001) and the use of antibiotics in livestock as a contributing factor to AMR (P = 0.01). Pharmacy personnel had better attitudes towards AMR as a public health problem (P = 0.001) and the use of antibiotics in livestock as a source of resistant pathogens (P = 001). Lastly, more pharmacy personnel than nurses participated in awareness campaigns (P = 0.029), continued professional development (P = 0.001) and courses on the use of antibiotics and AMR (P = 0.028). Conclusions The study showed that most participants had adequate knowledge, a positive attitude and good practices towards AMR. Significant differences in knowledge, attitudes and practices were observed between pharmacy personnel and nurses in AMR, highlighting a need for increased educational programmes for these healthcare personnel.