Objective To investigate bacterial infection in laboratory rats and mice from production facilities in Shanghai. Methods Programs for monitoring bacteria in laboratory animals were compared between China and elsewhere. A total of 848 samples were collected from the laboratory animal production facilities of Shanghai from 2014 to 2016. Helicobacter spp. , C. rodentium, C. bovis, S. aureus, P. aeruginosa, and K. pneumoniae were tested by PCR for the monitoring project. Results S. aureus contamination was not identified in SPF facilities in 2014 and 2016. K. pneumoniae contamination was not identified in clean level facilities in 2014. There were different degrees of contamination by other pathogens in facilities of different levels. Helicobacter spp. and C. bovis could be detected in all clean level facilities. Helicobacter spp. , C. rodentium, and C. bovis were detected in animals at all levels. The rates of positivity for Helicobacter spp. and P. aeruginosa declined over time, but those of C. rodentium, C. bovis, and K. pneumoniae rose. Conclusions This study investigated the prevalence of bacteria in rats and mice in Shanghai from 2014 to 2016. This work should promote the quality of laboratory animals and the levels of feeding and management, and also provide an important reference for the optimization of standards for laboratory animals.