Abstract Background: Healthcare Workers (HCW) are repeatedly exposed to SARS-CoV-2 infection. The aim of this study was to identify factors associated with SARS-CoV-2 infection among HCW in one of the largest cities in Colombia. Methods: We conducted a case-control study, where cases had a positive reverse transcription-polymerase chain reaction and controls had a negative result. Participants were randomly selected and interviewed by phone. Analyses were performed using logistic regression models. Results: A total of 110 cases and 113 controls were included. Men (AdjOR 4.1 95%CI 1.7-10), Nurses (AdjOR 11.2 95%CI 1.1-119.6), not using a high-performance filtering mask (AdjOR 2.2 95%CI 1-5.1) and inadequate use of personal protective equipment (AdjOR 4.8 95%CI 1.1-19.6) were identified as risk factors. Conversely, graduate (AdjOR 0.1 95%CI 0.01-0.5) and postgraduate (AdjOR 0.05 95%CI 0.004-0.5) education, feeling scared or nervous (AdjOR 0.5 95%CI0.2-0.9), not wearing any of gloves, hat and googles/face shields (AdjOR 0.1 95%CI0.02-0.4), and the use of high-performance filtering or a combination of fabric plus surgical mask (AdjOR 0.2 95%CI 0.1-0.8) outside the workplace were protective factors. Conclusion: This study highlights the protection provided by high-performance filtering mask or double masking among HCW. Individual factors and the difficulties of wearing other protective equipment needs to be considered in designing, implementing and monitoring COVID-19 biosafety protocols for HCW.