Objective To study the pathological spectrum of neonatal sepsis and antibiotic resistance of bacteria and fungi isolated from neonatal blood samples. Methods Clinical information of neonates with sepsis admitted to neonatal ward of our Hospital were retrospectively collected from January 2007 to December 2014 and analyzed. Results During the study period, a total of 23 224 infants were admitted to neonatal ward. Among them, 608 were diagnosed with sepsis (2. 62% , 608 / 23 224). Blood culture samples were positive in 412 cases, the rest (196 cases) were diagnosed according to clinical manifestations and laboratory tests. The composition ratio of sepsis during the first 4 years was significantly lower than that of the subsequent 4 years (2. 58% , 225 / 9 805 vs. 2. 85% , 383 / 13 419, P < 0. 05). Of 412 bacterial strains isolated, gram-positive bacteria, gram-negative bacteria and fungi accounted for 62. 38% , 30. 34% and 7. 28% , respectively. For the 155 strains isolated during first 4 years, the number of G + bacteria, G - bacteria and fungi were 106 strains (68. 39% ), 40 strains (25. 81% ), 9 strains ( 5. 81% ), respectively. The coagulase-negative staphylococcus ( CONS ) accounted for 90. 57% of G + strains. Klebsiella pneumoniae and Escherichia coli accounted for 50. 00%and 15. 00% of G - strains respectively. 257 strains were isolated during the subsequent 4 years, of which G + bacteria, G - bacteria and fungi accounted for 151 strains (58. 75% ), 85 strains (33. 07% ), 21 strains (8. 17% ), respectively. CONS accounted for 74. 83% of G + strains, Klebsiella pneumoniae and Escherichia coli accounted for 30. 58% and 35. 29% of G - strains. The composition ratio of CONS and Escherichia coli were significantly different between first and subsequent 4 years (P < 0. 05). G +bacteria were resistant to penicillin, oxacillin and macrolide, sensitive to piperacillin / tazobactam and quinolone. We did not identify any G + strain resistant to vancomycin. In general, G - bacteria were resistant to ampicillin and cephalosporins, sensitive to the beta lactamase inhibitor compound preparation and quinolones, and highly sensitive to carbapenems. Fungi generally were sensitive to conazoles. Conclusions Neonatal sepsis are mainly caused by CONS, Klebsiella pneumoniae and Escherichia coli also play important roles. G + bacteria and G - bacteria in general are resistant to penicillin and cephalosporins. All G + bacterial strains isolated from our cohort are sensitive to vancomycin. G - bacteria are generally sensitive to carbapenems. Fungi generally are sensitive to conazoles.