Objectives: The COVID-19 pandemic has globally affected healthcare workers’ (HCWs) health and wellbeing. Most studies on COVID-19 have focused on tertiary healthcare. The aim of this study was to increase the knowledge on the effects of the pandemic on working conditions in tertiary and primary healthcare. Material and Methods: The comparative cross-sectional study consisted of an online questionnaire sent to HCWs of the City of Helsinki (primary healthcare) and Helsinki University Hospital (tertiary healthcare). Altogether 1580 HCWs with direct patient contact participated in the study: 895 from tertiary and 685 from primary healthcare. Statistical analysis used SPSS 25 from IBM. The tests used were the χ² test, Fisher’s exact test, and binary logistic regression analysis. Results: Primary HCWs were less likely to treat COVID-19 patients (OR = 0.45, 95% CI: 0.37–0.56). However, both groups reported a similar number of COVID-19 infections, primary HCWs 4.9% and tertiary HCWs 5.0%, and workrelated quarantine was significantly more prevalent (OR = 1.96, 95% CI: 1.38–2.79) among primary HCWs. In addition, work-related wellbeing was poorer among primary HCWs than tertiary HCWs in terms of feeling more stressed at work (OR = 3.20, 95% CI: 2.55–4.02), not recovering from work (OR = 0.49, 95% CI: 0.39–0.62), reported mental wellbeing below normal levels (OR: 1.59, 95% CI: 1.26–2.00), and increased working hours (OR = 1.63, 95% CI: 1.25–2.12). Conclusions: The study demonstrates how the pandemic has affected the wellbeing and working conditions of not only tertiary but also less studied primary HCWs. The authors’ findings suggest that the challenges identified during the COVID-19 pandemic in the health and wellbeing of healthcare workers are even greater in primary care than in tertiary care. Int J Occup Med Environ Health. 2023;36(1):139–50