The COVID-19 pandemic interrupted routine healthcare services. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are often asymptomatic and therefore screening, on/post-treatment monitoring are required. Our aim was to determine the effect of the first, second, and third waves of the pandemic on HBV and HCV testing in Ontario, Canada. We extracted data from Public Health Ontario for HBV and HCV specimens from January 1, 2019 to May 31, 2021. Testing volumes were evaluated and stratified by age, sex, and region. Changes in testing volumes were analyzed by percent and absolute change. Testing volumes decreased in April 2020 with the first wave of the pandemic, and recovered to 72%-75% of pre-pandemic volumes by the end of the first wave. HBsAg testing decreased 33%, 18%, and 15%, and HBV DNA testing decreased 37%, 27%, and 20%, in each consecutive wave. Anti-HCV testing decreased 35%, 21%, and 19%, and HCV RNA testing decreased 44%, 30%, and 36%, in each consecutive wave. These trends were consistent by age, region, and sex. Prenatal HBV testing volumes were stable. In conclusion, significant decreases in HBV and HCV testing occurred during the first three waves of the pandemic and have not recovered. In addition to direct consequences on viral hepatitis elimination efforts, these data provide insight into the impacts of the pandemic on chronic disease screening and management in Ontario. Strategies to make up for missed testing will be critical to avoid additional consequences of COVID-19 long after the pandemic has resolved.