Introduction: This study aims to evaluate the pre-exposure and post-exposure efficacy of hydroxychloroquine prophylaxis among Healthcare Workers (HCW) to prevent COVID-19 infection. Materials and Methods: This was a retrospective cohort study. Pre-exposure prophylaxis was given to the HCW in the pre-exposure group according to their willingness. Post-exposure prophylaxis was initiated to HCW after exposure in the hospital or social area within 96 hours. The main outcome was symptomatic or asymptomatic COVID-19 infection confirmed by PCR during the study period for the pre-exposure prophylaxis group and symptomatic or asymptomatic COVID-19 infection confirmed by PCR within 5 to 14 days from exposure for the post-exposure prophylaxis group. An online structured survey was sent to HCW participating in the study. The data were also validated by the available medical records of the hospital and analyzed with R, an open-source statistical package. Results: A total of 492 HCW who worked between March 20 and June 20 of 2020 in our hospital were recruited for the study. A total of 40 (8.1%), 152 (31%), 266 (54%), and 34 (6.9%) HCWs had received pre-exposure prophylaxis, post-exposure prophylaxis, no-prophylaxis, or both pre and post-exposure prophylaxis, respectively. Eventually, 47 HCWs obtained a diagnosis of COVID-19 confirmed with the PCR test. The rate of COVID-19 was 9.6% among the HCW who participated in the study. PCR-confirmation rates of COVID-19 were 18%, 4.6%, 12%, and 2.9% in those receiving pre-exposure prophylaxis, post-exposure prophylaxis, no prophylaxis, and both pre-exposure and post-exposure prophylaxis, respectively. The final logistic model revealed a negative association between post-exposure prophylaxis and PCR confirmation COVID-19 disease (effect estimate: -1.179; 95% limits: -1.969-0.390). Conclusions: This study implies that pre-exposure prophylaxis is not protective. Post-exposure prophylaxis with hydroxychloroquine may decrease the acquisition of COVID-19 disease.