The uptake of COVID-19 vaccines is critical to address the severe consequences of the disease. Previous studies have suggested that many healthcare workers (HCWs) are hesitant to receive the COVID-19 vaccine, further enhancing hesitancy rates within countries. COVID-19 vaccine acceptance and hesitancy levels are currently unknown among HCWs in Zambia, which is a concern given the burden of infectious diseases in the country. Consequently, this study assessed COVID-19 vaccine acceptance and hesitancy among HCWs in Lusaka, Zambia. A cross-sectional study was conducted among 240 HCWs between August and September 2022, using a semi-structured questionnaire. Multivariable analysis was used to determine the key factors associated with vaccine hesitancy among HCWs. Of the 240 HCWs who participated, 54.2% were females. A total of 72.1% of the HCWs would accept being vaccinated, while 27.9% were hesitant. Moreover, 93.3% of HCWs had positive attitudes towards COVID-19 vaccines, with medical doctors having the highest mean attitude score (82%). Encouragingly, HCWs with positive attitudes towards COVID-19 vaccines had reduced odds of being hesitant (AOR = 0.02, 95% CI: 0.01–0.11, p < 0.001). Overall, acceptance of the COVID-19 vaccine among HCWs in Lusaka, Zambia, was high, especially by those with positive attitudes. However, the current hesitancy among some HCWs is a concern. Consequently, there is a need to address this and encourage HCWs to fully promote vaccination programs going forward.