Objective To explore whether there is any difference between the simplified two-step hygienic hand disinfection and the six-step hygienic hand disinfection method recommended in the WHO guidelines for hand hygiene in health care. Methods 32 clinical health care workers (HCWs) in a tertiary general hospital were grouped randomly by randomized controlled cross-over trial, two-step method was adopted in trial group and six-step method was adopted in control group, trial was crossly conducted with each other between two periods, with an interval of one day, the average killing logarithm of natural bacteria on hands of two groups of HCWs before and after hygienic hand disinfection was compared. Results The average killing logarithm values after HCWs' hygienic hand disinfection in trial group and control group were both >1, disinfection effect were both qualified, the decrease of colony number after hygienic hand disinfection of HCWs in two groups were both statistically significant (both < 0.001); there was no significant difference in the average killing logarithm of bacteria between two kinds of hygienic hand disinfection (P=0.973). Conclusion The effect of two-step hygienic hand disinfection is the same as that of six-step hygienic hand disinfection, two-step method takes less time and has higher efficiency, which is worthy of clinical promotion.