Objective To understand the impact of bed-making manipulation on the air surrounding bed units in hemodialysis room, evaluate the effectiveness of routine terminal disinfection, and provide scientific basis for optimizing infection control measures. Methods Air specimens (pre-bed-making group) and environmental object surface specimens (pre-terminal disinfection group) around bed units were collected when hemodialysis was about to be fi-nished. Air specimens after bed-making (bed-making group) and environmental object surface specimens after terminal disinfection (terminal disinfection group) were also collected. Bacterial colonies were counted and identified. Results A total of 714 air specimens were collected from 238 bed units of 45 hemodialysis units before and during bed-making. The average bacterial colony count during bed-making was higher than that before bed-making ([2.72±3.43] CFU/plate vs [0.69±1.50] CFU/plate, P < 0.05). The qualified rate of microbial colony count before bed-making was higher than that during bed-making (96.64% vs 64.71%, P < 0.05). A total of 450 environmental object surface specimens from 90 bed units of 45 hemodialysis units were collected before and after terminal disinfection. The average bacterial colony count before terminal disinfection was higher than that after terminal disinfection ([8.18±20.6] CFU/cm² vs [1.49±3.44] CFU/cm², P < 0.05). The qualified rate of microbial colony count after terminal disinfection was higher than that before terminal disinfection (98.22% vs 92.44%, P < 0.05). The microorganisms in the air and environment were mainly coagulase negative Staphylococcus. Conclusion Bed-making has a certain impact on the air microorganisms around the bed units, and terminal disinfection can reduce the bacterial co-lony count of the environment. It is necessary to pay attention to the cleaning and disinfection of the environment for medical treatment, and carry out bed-making manipulation under clear condition to minimize the risk of infection.