Objective: To understand the knowledge of disinfection and its influencing factors among caregivers in childcare centers in Huangpu District, Shanghai, in order to provide a basis for the future development of targeted training programs and the work plan to enhance the professional level of disinfection practitioners in childcare centers. Methods: A total of 423 caregivers from 62 childcare centers (including nursery schools) in Huangpu District were selected for a questionnaire about disinfection knowledge, influencing factors, and training needs in March 2023. Differences in disinfection knowledge among subjects with different characteristics were compared using χ 2 tests, and influencing factors were analyzed using a multi factor binary Logistic regression model. Results: The overall knowledge rate of disinfection among caregivers was 50.12%, and those in public kindergartens, private ones, and nursery schools were 51.35%, 46.18%, and 42.57%, respectively, with statistically significant differences ( χ 2=14.25, P < 0.05 ). The caregivers in the highest level kindergartens ( OR =4.50, 95% CI =1.97-10.29), in first level ones ( OR =4.29, 95% CI = 1.98-9.33), in the institutions had clusters of outbreaks ( OR =1.87, 95% CI =1.14-3.07), in which the number of children to caregivers ratio being less than 10∶1 ( OR =21.81, 95% CI =2.55-186.59), with 6-14 years of working experience ( OR =3.51, 95% CI = 1.59 -7.75) had better knowledge of disinfection( P <0.05). Conclusions Knowledge of disinfection among caregivers of childcare institutions is low in Huangpu District, Shanghai. Training of caregivers disinfection knowledge should be strengthened for caregivers with fewer years of experience, in childcare institutions, to improve caregivers disinfection expertise and skills.