Objective To understand the detection and antimicrobial resistance of bacteria in the pharynx and umbilicus of neonates during admission to hospital, and provide basis for the prevention and control of neonatal infection. Methods Active screening results of 3 639 neonates admitted to the neonatal ward of a hospital from 2017 to 2022 were retrospectively investigated. Changes in bacterial detection in the three years (2017-2019) before COVID -19 pandemic and the three years (2020-2022) during COVID -19 pandemic were compared and analyzed. Results From 2017 to 2022, a total of 3 639 neonates were investigated, and 1 152 strains were detected from 914 neonates. The top 5 detected bacteria were Escherichia coli, Staphylococcus aureus, coagulase negative Staphylococcus, Enterococcus faecalis, and Klebsiella pneumoniae. The top 3 bacteria detected in the three years before the pandemic were different from that during the pandemic, and the positive rate of bacteria detected in umbilicus was higher than that in pharynx, with statistically significant differances (P < 0.001). The major multidrug-resistant organism was methicillin-resistant Staphylococcus aureus (MRSA) (n=71). The positive detection rates of Staphylococcus aureus and MRSA in the three years during COVID -19 epidemic were both significantly lower than that before the pandemic (2.79% vs 4.76%; 0.63% vs 1.31%, respectively, both P < 0.05). The positive rate of MRSA in umbilicus was higher than that in pharynx (2.25% vs 0.38%) in the three years before pandemic. The positive detection rate of MRSA in umbilicus in the three years during the epidemic decreased compared to before the pandemic (0.61% vs 2.25%)(P < 0.001). Antimicrobial usage rate of hospitalized patients from the neonatal ward in the three years during the epidemic was lower than that before the pandemic (60.89% vs 67.42%, P < 0.001). Conclusion The positive rates of Staphylococcus aureus and MRSA detected in umbilical via neonatal screening were lower during the COVID -19 epidemic than that before the pandemic, which may be related to the increased awareness of public health behaviors in and outside hospitals during the epidemic.