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Background Healthcare workers (HCWs) and other essential workers are at risk of occupational infection during the COVID-19 pandemic. Several infection control strategies have been implemented. Evidence shows that universal masking can mitigate COVID-19 infection, though existing research is limited by secular trend bias. Aims To investigate the effect of hospital universal masking on COVID-19 incidence among HCWs compared to the general population. Methods We compared the 7-day average incidence rates between a Massachusetts (USA) healthcare system and Massachusetts residents statewide. The study period was from 17 March (the date of first incident case in the healthcare system) to 6 May (the date Massachusetts implemented public masking). The healthcare system implemented universal masking on 26 March, we allotted a 5-day lag for effect onset and peak COVID-19 incidence in Massachusetts was 20 April. Thus, we categorized 17–31 March as the pre-intervention phase, 1–20 April the intervention phase and 21 April to 6 May the epidemic decline phase. Temporal incidence trends (i.e. 7-day average slopes) were compared using standardized coefficients from linear regression models. Results The standardized coefficients were similar between the healthcare system and the state in both the pre-intervention and epidemic decline phases. During the intervention phase, the healthcare system's epidemic slope became negative (standardized β: −0.68, 95% CI: −1.06 to −0.31), while Massachusetts' slope remained positive (standardized β: 0.99, 95% CI: 0.94 to 1.05). Conclusions Universal masking was associated with a decreasing COVID-19 incidence trend among HCWs, while the infection rate continued to rise in the surrounding community. [ABSTRACT FROM AUTHOR]