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 4.13 95% CI 1.70–10.05), Nurses (AdjOR 11.24 95% CI 1.05–119.63), not using a high-performance filtering mask (AdjOR 2.27 95% CI 1.02–5.05) and inadequate use of personal protective equipment (AdjOR 4.82 95% CI 1.18–19.65) were identified as risk factors. Conversely, graduate (AdjOR 0.06 95% CI 0.01–0.53) and postgraduate (AdjOR 0.05 95% CI 0.005–0.7) education, feeling scared or nervous (AdjOR 0.45 95% CI 0.22–0.91), not always wearing any gloves, caps and goggles/face shields (AdjOR 0.10 95% CI 0.02–0.41), and the use of high-performance filtering or a combination of fabric plus surgical mask (AdjOR 0.27 95% CI 0.09–0.80) outside the workplace were protective factors. Conclusion This study highlights the protection provided by high-performance filtering masks or double masking among HCW. Modifiable and non-modifiable factors and the difficulty of wearing other protective equipment needs to be considered in designing, implementing and monitoring COVID-19 biosafety protocols for HCW.