This single-center study aimed to explore the factors associated with coronavirus disease (COVID-19) transmission in a hospital. All laboratory-confirmed COVID-19 cases among health care workers (HCWs) in a tertiary hospital in Malaysia were analyzed cross-sectionally from January 25, 2020, to September 10, 2021. A total of 897 HCWs in the hospital had laboratory-confirmed COVID-19 infection during the study period. Around 37.4% of HCWs were suspected to acquire COVID-19 infection from the hospital workplace. Factors associated with lower odds of workplace COVID-19 transmission were being females, ≥30 years old, fully vaccinated, and working as clinical support staff. Involvement in COVID-19 patient care was significantly associated with higher odds (adjusted odds ratio = 3.53, 95% confidence interval: [2.42, 5.12]) of workplace COVID-19 transmission as compared with non-workplace transmission. Most HCWs in the tertiary hospital acquired COVID-19 infection from non-workplace settings. During a pandemic, it is important to communicate with HCWs about the risk of both workplace and non-workplace COVID-19 transmission and to implement measures to reduce both workplace and non-workplace COVID-19 transmission.