Background: Healthcare workers have a higher risk of SARS CoV2 infection with implications for transmission of infection and the safety of workers and patients. Objective: To assess knowledge on COVID-19 and the safety practices among selected healthcare workers in southwest Nigeria. Methods: A cross-sectional study of 210 workers providing direct care to patients during the COVID-19 pandemic was conducted. Respondents were recruited through simple random sampling of members of online platforms of healthcare workers in Osun, Ondo and Ekiti States. Information on sociodemographic characteristics, knowledge of PPE and safety practices was obtained through a close-ended questionnaire. Results: The mean age of the respondents was 36.5±7.5 years. About 29% and 30% of respondents were from the State and Federal Government-owned Teaching Hospitals, respectively. A little above half (58.1%) had good knowledge of COVID-19, while 62.1% used PPE always when attending to suspected COVID19 cases. More than half (53.8%) had been trained on infection prevention and control (IPC), but only 34.3% adhered to good safety practices. Healthcare workers in State government-owned teaching hospitals had lower odds of good safety practices than those in Federal Teaching Hospitals (OR = 0.42, 95% CI = 0.19-0.93, p = 0.031). Conclusion: The knowledge of appropriate PPE and practice of safety precautions among healthcare workers is sub-optimal. This may predispose to increased COVID-19 transmission among healthcare workers, patients, and their families. Training and retraining healthcare workers, especially those from hospitals identified by the study as having poor safety practices, should be encouraged.