BACKGROUND: This study assesses the perceptions and acceptance of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination. It also examines its influencing factors among the healthcare workers (HCWs) in the Eastern Cape, South Africa. METHODS: In this cross-sectional study performed in November and December 2020, a total of 1308 HCWs from two large academic hospitals participated in the Eastern Cape Healthcare Workers Acquisition of SARS-CoV-2 (ECHAS) study. Validated measures of vaccine hesitancy were explored using a questionnaire. Logistic regression was used to identify the determinants of vaccine hesitancy. RESULTS: The majority were nurses (45.2%), and at risk for unfavourable Covid-19 outcome, due to obesity (62.9%) and having direct contact with individuals confirmed to have Covid-19 (77.1%). The overall acceptance of SARS-CoV-2 vaccine was 90.1%, which differed significantly by level of education. Individuals with lower educational attainment (primary and secondary education) and those with prior vaccine refusal were less likely to accept the SARS-CoV-2 vaccine. However, positive perceptions about the SARS-CoV-2 vaccine were independently associated with vaccine acceptance. CONCLUSIONS: The high level of acceptance of SARS-CoV-2 vaccine is reassuring; however, HCWs with a lower level of education and those with prior vaccine refusal should be targeted for further engagements to address their concerns and fears.