Although multilayered strategies including preventive behaviors should be adopted to mitigate coronavirus disease 2019 (COVID-19) transmission, evidence on the effectiveness of preventive behaviors against COVID-19 remains limited. This Internet-based prospective cohort study collected baseline data in November 2020 and follow-up data in February 2021, during the third wave of the epidemic in Japan. Among the 19 941 included participants, the percentages reporting that they always used a face mask, practiced hand washing/disinfection, gargling, and ensuring proper room ventilation were 85.4%, 36.0%, 51.1%, and 44.6%, respectively. Multiple logistic regression analyses revealed that less frequently practicing hand washing/disinfection (odds ratio [OR] = 1.20, 95% confidence interval [CI]: 1.10-1.32), gargling (OR = 1.20, 95% CI: 1.10-1.30), and ensuring proper room ventilation (OR = 1.38, 95% CI: 1.26-1.52) were significantly associated with self-reported COVID-19-like illness (CLI). These results suggest that personal preventive behaviors may be effective in reducing CLI, even when universal masking is practiced.