Objective To investigate the microbial contamination and management of refrigerators in the ward treatment rooms of a tertiary first-class hospital, and provide reference for strengthening the cleaning and disinfection of medical refrigerators in the ward treatment rooms. Methods A total of 40 refrigerators in use from the treatment rooms of different wards in the hospital were randomly selected for microbial sampling, culture, and bacterial identification. Forty nurses were randomly chosen from the corresponding wards for a questionnaire survey on the daily management of refrigerators. Results A total of 223 specimens were collected, with 142 microbial positive specimens and a positive rate of 63.68%. A total of 247 bacterial strains were detected, including 41.30% (n=102) Gram-positive bacteria, 10.93% (n=27) Gram-negative bacteria, and 47.77% (n=118) fungi. Two strains of multidrug-resistant Pseudomonas aeruginosa from the bottom and drainage tank of the same refrigerator from class-Ⅲ environment were detected, with a detection of multidrug-resistant organisms being 0.90% (2/223). The frequency and methods of routine cleaning and disinfection of refrigerators varied among different wards. Conclusion There are deficiencies in the cleaning and disinfection management of refrigerators in ward treatment rooms of medical institutions. More attention should be paid to the cleaning and disinfection of refrigerators in wards. The healthcare-associated infection management departments should strengthen corresponding supervision and management.