Objective To explore the key contents of nosocomial infection prevention and control training for medical staff in secondary and above hospitals, so as to provide scientific basis for training in the future. Methods The medical workers who participated in the training of nosocomial infection prevention and control in secondary and above hospitals of 6 prefecture-level cities in Jiangxi province in December 2020 were selected. The same questionnaire was used to test the participants before and after training, and the changes of scores before and after training were compared. Results A total of 73 medical workers were included. After training, the total scores of the questionnaire (14.13±1.95 vs. 11.27±2.76; t=11.053, P<0.001), scores of manual hygiene specifications knowledge unit (4.63±0.65 vs. 4.02±1.37; t=4.215, P<0.001), scores of hospital isolation technical specifications knowledge unit (4.28±1.05 vs. 3.47±1.29; t=4.895, P<0.001), scores of airborne disease hospital infection prevention and control norms knowledge unit (5.21±0.96 vs. 3.76±1.04; t=10.419, P<0.001) and the overall accuracy of the questionnaire (83.00% vs. 66.32%) were higher than those before training. Conclusions After the training, the accuracies of different topics are improved, but there is still room for improvement in each knowledge unit. More effective training strategies should be considered.