IntroductionOur aim was to gain insight into the effect of COVID-19 measures on SARS-CoV-2 incidence in secondary schools and the association with classroom CO2concentration and airborne contamination.MethodsBetween October 2020—June 2021, 18 schools weekly reported SARS-CoV-2 incidence and completed surveys on school-initiated COVID-19 measures (e.g. improving hygiene or minimizing contacts). CO2was measured in occupied classrooms twice, and SARS-CoV-2 air contamination longitudinally using electrostatic dust collectors (EDC) and analyzed using RT-qPCR. National COVID-19 policy measures varied during pre-lockdown, lockdown and post-lockdown periods. During the entire study, schools were recommended to improve ventilation. SARS-CoV-2 incidence rate ratios (IRR) were estimated by Generalized Estimating Equation (GEE) models.ResultsDuring 18 weeks follow-up (range: 10–22) SARS-CoV-2 school-incidence decreased during national lockdown (adjusted IRR: 0.41, 95%CI: 0.21–0.80) and post-lockdown (IRR: 0.60, 0.39–0.93) compared to pre-lockdown. School-initiated COVID-19 measures had no additional effect. Pre-lockdown, IRRs per 10% increase in time CO2exceeded 400, 550 and 800 ppm above outdoor level respectively, were 1.08 (1.00–1.16), 1.10 (1.02–1.19), and 1.08 (0.95–1.22). Post-lockdown, CO2-concentrations were considerably lower and not associated with SARS-CoV-2 incidence. No SARS-CoV-2 RNA was detected in any of the EDC samples.ConclusionDuring a period with low SARS-CoV-2 population immunity and increased attention to ventilation, with CO2levels most of the time below acceptable thresholds, only the national policy during and post-lockdown of reduced class-occupancy, stringent quarantine, and contact testing reduced SARS-CoV-2 incidence in Dutch secondary schools. Widespread SARS-CoV-2 air contamination could not be demonstrated in schools under the prevailing conditions during the study.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12889-023-16146-0.