Objective To investigate the incidence and adverse prognosis of late onset sepsis (LOS) in neonates in neonatal intensive care unit (NICU). Methods A retrospective study was conducted to collect and analyze the perinatal condition, underlying diseases, invasive procedures, and adverse prognosis of neonates in NICU of a regional maternal and child healthcare hospital from 2019 to 2023. According to whether LOS occurred during hospitalization, neonates were divided into LOS group and non-LOS group. The LOS group was divided into 5 subgroups based on whether invasive procedures were performed: LOS plus umbilical vein catheter (UVC) group, LOS plus peripherally inserted central catheter (PICC) group, LOS plus sequential catheter group, LOS plus tracheal intubation group, and LOS plus lumbar puncture group, the relationship between LOS and adverse prognosis was analyzed. Results Among 2 945 neonates in NICU, 354 (12.02%) developed LOS. Comparison between LOS groups and non-LOS group were as follows: in term of perinatal condition of neonates, there were statistically significant difference in weight, gestational age, and whether they were twins between the two groups (all P<0.001); in term of underlying diseases, there were statistically significant differences in the number of cases of maternal gestational hypertension, neonatal asphyxia, neonatal congenital heart disease, neonatal ventricular dilation, neonatal pneumonia, neonatal hyperthyrotropinemia, and neonatal anemia, as well as five invasive procedures between the two groups (all P<0.05). Compared with the non-LOS group, the incidences of retinopathy of prematurity (ROP), neonatal necrotizing enterocolitis (NNEC), bronchopulmonary dysplasia (BPD), and neonatal respiratory distress syndrome (NRDS) in LOS group were all higher (all P<0.001). Regression analysis showed that compared with the non-LOS groups, the risk of ROP increased in the LOS group and its subgroups, with the LOS plus sequential catheter group having a 2.27-fold higher risk of ROP than non-LOS group; the risk of NNEC increased in the LOS group and its subgroups, with the LOS plus UVC group having an 8.29-fold higher risk of NNEC than the non-LOS group. Except for the LOS plus UVC group, the risk of BPD increased in the LOS group and other subgroups, with the LOS plus PICC group and LOS plus sequential catheter group having 4.68- and 4.64-fold higher risk of BPD than the non-LOS group, respectively; the risk of NRDS in the LOS plus PICC group was 6.84-fold higher than the non-LOS group (all P<0.05). The top three pathogens causing LOS were coagulase negative Staphylococcus, Klebsiella pneumoniae, and Escherichia coli. Conclusion LOS can significantly increase the risks of ROP, NNEC, BPD, and NRDS. LOS plus invasive procedures can further increase the risk of adverse prognosis.