Objective To observe the effect of multi-modal hand hygiene (HH) intervention on HH compliance, as well as the relationship between HH compliance and the healthcare-associated (HA) case infection incidence. Methods From 2014 to 2022, the infection control team in a tertiary first-class hospital implemented multi-modal HH intervention for health care workers (HCWs). The changing trend of HH monitoring data, the correlation between HH compliance rate and HA case infection incidence were analyzed retrospectively. Results The consumption of HH products in the wards showed a stable upward trend; HH compliance rate increased from 64.98% in 2014 to 85.01% in 2022 (P<0.001), and HA case infection incidence decreased from 1.21% to 0.83% (P<0.05). HH compliance rate was negatively correlated with HA case infection incidence (r=-0.369, P=0.027). HH compliance rates in different regions and job posts in each quarter were increased (P<0.001). For 5 different HH moments in each quarter, HH compliance rate fluctuated slightly before sterile manipulation and after touching patient; presented rising trend after touching surroundings around patient, and decreased before touching patient and after touching patient's body fluid since 2020 (P<0.001). Conclusion Multi-modal HH intervention can improve the HH compliance of HCWs, improving their HH awareness is conducive to reducing HA case infection incidence.