ObjectivesTo assess whether pneumococcal nasopharyngeal carriage among children aged 24–60 months reduced during the coronavirus disease 2019 (COVID-19) pandemic in Novi Sad, Serbia, and to investigate the overall prevalence of carriage, serotype distribution and dominant serotypes 2–3 years after the introduction of pneumococcal conjugate vaccine 10. Design and methodsThis prospective, observational study was conducted in February–March 2020, September–November 2020 and April–June 2021, enabling the comparison of results in the pre-pandemic/early pandemic period with two periods during the COVID-19 pandemic. Pneumococci were identified by standard microbiological methods. Serotype identification was performed using conventional multiplex polymerase chain reaction assays. ResultsAmong 1623 children tested, 515 (31.7%, 95% confidence interval 29.4–34.0%) carried pneumococci. A significant increase in prevalence was found between February–March 2020 and September–November 2020 (P=0.0085), with no difference found between September–November 2020 and April–June 2021 (P=0.0524). Pneumococcal colonization was significantly higher in children who were fully vaccinated and among children who attended day care centres. The dominant serotypes were 15B, 6B, 19F, 11A, 6C, 6A, 3, 23F and 19A, representing 66.4% of all isolates. ConclusionsThis study found that pneumococcal nasopharyngeal carriage in children aged 24–60 months was high before the COVID-19 pandemic, and then increased during the pandemic. This rules out a major role of COVID-19 in the suppression of carriage and, probably, transmission.