Introduction: Health care workers (HCWs) are at a high risk of infection owing to occupational exposure to patients and virus-contaminated surfaces. Objectives: The study was aimed to reveal and compare the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among patient-facing HCWs across 3 dental clinics equipped with different types of aspirating systems. Methods: This retrospective cohort study included 157 HCWs (43.58 ± 1.66 y) from 3 dental clinics in Ekaterinburg (Russian Federation) who reported to work during the coronavirus disease pandemic. All HCWs underwent serological testing once a week to detect immunoglobulin G and M antibodies against the SARS-CoV-2. The V6000 aspirating system with a vacuum controller (dry or semidry mode) and high-efficiency particulate air (HEPA) filters was used at clinics A and B, and the aspirated aerosol and air were evacuated and dissipated into the atmosphere. The VS900 aspirating vacuum pump without HEPA filters was used at clinic C. The aspirated aerosol and air were evacuated and dissipated into the operatories. All dental clinics followed the same recommendations for dental patient management and types of personal protective equipment used. Results: The estimated prevalence of SARS-CoV-2 infection was 11.5% (19 HCWs) over a 5-mo follow-up (May to August 2020). The prevalence of infection was unaffected by sex or the role of the member in the dental team (dentist/dental assistant). The prevalence of SARS-CoV-2 infection (+) was significantly higher at clinic C (equipped with an aspirating vacuum pump without HEPA filters) than at other clinics. Conclusion: The type of aspirating system used and the presence of HEPA filters could affect the prevalence of SARS-CoV-2 infection across dental clinics. Therefore, we recommend the use of aspirating systems installed with HEPA filters, which evacuate and dissipate aerosols into specialized areas. Knowledge Transfer Statement: This report confirms that dentists, being patient-facing HCWs, are at a high risk of acquiring the SARS-CoV-2 infection and identifies gaps in the protection of patients and staff in dental settings. © International & American Associations for Dental Research 2021.