Objective To compare germicidal effect of three disinfectants acting on frequently-touched surfaces in Intensive Care Unit (ICU) at different time points after disinfection so as to put forward the reasonable disinfection method and interval before the next disinfection. Methods We wiped the four frequently-touched surfaces in ICU with disinfectant containing acidic electrolyzed oxidizing water (EOW) from the building system of hospital, disinfectant wipes, and 500 mg/L chlorine respectively. The culture samples were collected from the surfaces before wiping, and 10 minutes, 30 minutes, 1 hour, 2 hours and 4 hours after wiping respectively. The bacterial clearance rate and the qualified rate of bacterial colony counts on the surfaces were compared among the three different disinfectants at different time points after disinfection. Results The bacterial killing rate of three methods for disinfection of object surfaces decreased with the passing of time. The bacterial killing rate of EOW from the building system of hospital was lower than that of the other two methods at all five time points after disinfection (P< 0.05). The bacterial killing rate at hour four after disinfection using chlorine-containing disinfectant and disinfectant wipes was higher than 90.0%. The qualified rate of bacterial colony counts on the surfaces at 10 and 30 minutes after disinfection among the three groups was not significantly different (P>0.05). The qualified rate of bacterial colony counts on the surfaces disinfected by EOW from the building system of hospital was lower than that in the other two groups at the other three time points (P<0.05), and it was totally unqualified at hour four after disinfection. Conclusions The germicidal effect of EOW from the building system of hospital is inferior to chlorine disinfectant and disinfectant wipes. Moreover, the surface can be easily recontaminated after disinfection. It is suggested that EOW should be used in ICU every other hour. and the other two disinfection methods should be used every two hours.