Objective To quantitatively analyze the impact of environmental hygiene on the occurrence of healthcare-associated infections (HAI). Methods Monitoring data of HAI and environmental hygiene from a tertiary first-class hospital from January 2018 to December 2022 were collected, and the impact of environmental bacterial colony forming unit (CFU) on the occurrence of HAI was analyzed by a time-series generalized additive model. Results The single-contamination model showed a significant positive correlation between HAI and staff's hand bacterial CFU (β₁=0.009, P=0.012). For an increase of 1 interquartile range (IQR) in the monthly mean CFU per dish (MCFU/Dish) of staffs' hand, the incidence of HAI increased by 13.28% (95%CI: 2.82%-24.81%). Subgroup and lag effect analysis showed that when the monthly MCFU/Dish (after hand disinfection) of staffs' hand increased by one IQR, the excess risk (ER) of HAI for the month (lag0) was 16.26% (95%CI: 15.45%-17.09%). In the multi-contamination model, the correlation between surface contamination and HAI was also statistically significant. Conclusion There is a significant correlation between hospital environmental hygiene and the occurrence of HAI.