My 5 moments (M5M) was used less frequently among cleaning staff members, suggesting that a poor compliance score in this group may not indicate deficient handwashing. This quasi-experimental study compared hand hygiene compliance (HHC), hand hygiene (HH) moments, and HH time distribution in the control group (no HH intervention;n= 21), case group 1 (normal M5M intervention;n= 26), case group 2 (extensive novel six moments (NSM) training;n= 24), and case group 3 (refined NSM training;n= 18). The intervention’s effect was evaluated after 3 months. The HHC gap among the four groups gradually increased in the second intervention month (control group, 31.43%; case group 1, 38.74%; case group 2, 40.19%; case group 3, 52.21%;p< 0.05). After the intervention period, the HHC of case groups 2 and 3 improved significantly from the baseline (23.85% vs. 59.22%, 27.41% vs. 83.62%, respectively;p< 0.05). ‘After transferring medical waste from the site’ had the highest HHC in case group 3, 90.72% (95% confidence interval, 0.1926–0.3967). HH peak hours were from 6 AM to 9 AM and 2 PM to 3 PM. The study showed that the implementation of an NSM practice can serve as an HHC monitoring indicator and direct relevant training interventions to improve HH among hospital cleaning staff.