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• Reducing the transmission from asymptomatic and presymptomatic patients is critical in controlling of severe acute respiratory syndrome coronavirus 2 circulation. • Screening and isolation of asymptomatic health care workers using RT-PCR significantly reduces hospital absenteeism. • The reduction of virus circulation showed also a trend toward of reduction in the transmission of the infection to hospitalized patients. Reducing the transmission of SARS-CoV-2 from asymptomatic and pre-symptomatic patients is critical in controlling the circulation of the virus. This study evaluated the prevalence of Reverse transcription polymerase chain reaction (RT-PCR) positivity in serial tests in 429 asymptomatic health care workers (HCW) and its impact on absenteeism. HCW from a COVID-19 reference hospital were tested, screened, and placed on leave. A time-series segmented regression of weekly absenteeism rates was used, and cases of infection among hospitalized patients were analyzed. Viral gene sequencing and phylogenetic analysis were performed on samples from HCW who had a positive result. A significant decrease in absenteeism was detected 3–4 weeks after the intervention at a time of increased transmission within the city. The prevalence of RT-PCR positivity among asymptomatic professionals was 17.3%. Phylogenetic analyses (59 samples) detected nine clusters, two of them strongly suggestive of intrahospital transmission with strains (75% B.1.1.28) circulating in the region during this period. Testing and placing asymptomatic professionals on leave contributed to control strategy for COVID-19 transmission in the hospital environment, and in reducing positivity and absenteeism, which directly influences the quality of care and exposes professionals to an extra load of stress. [ABSTRACT FROM AUTHOR]