Objective To compare the epidemic status of nosocomial infections (NIs) among medical institutions at different levels. Methods The cross-sectional surveys on prevalence rates of NIs, distribution of NIs, and antimicrobial use were conducted through combination of bedside investigation and medical record reviewing among all in-patients of 20 medical institutions in Baoshan District, Shanghai from 00:01 to 24:00 on November 12th 2014, December 9th 2015, and November 30th 2016, respectively. Results A total of 18 762 patients were investigated, the prevalence rate of NIs in the first, second, and third class hospitals were 5.36%, 2.37%, 1.68%, respectively (χ2=88.497, P<0.05). The main NIs sites were lower respiratory tract, urinary tract, and upper respiratory tract in the first and second grade hospitals; while were other unclassified sites, respiratory tract, and upper respiratory tract in the third grade hospitals. The utilization rates for antimicrobial in the first, second, and third grade hospitals were 5.88%, 31.64%, and 42.11%, respectively (χ2=928.148, P<0.05); submission rates for specimen were 9.82%, 48.89%, and 82.39%, respectively (χ2=601.347, P<0.05). Four cases of pathogen were reported in the first grade hospitals, 94 in the second grade hospitals, and 96 in the third grade hospitals. The in-patients in different hospitals with different genders, ages, and departments had a statistical difference in prevalence rate of NIs (P<0.05) . Conclusion The first grade hospitals need to enhance the etiological examination; the third grade hospitals should severely restrict the antimicrobial utilization, and refine the prevention and control work for NIs.