Background Neonatal infection refers to the infection of the newborn during the first twenty-eight days of life. It is one of the causes of infant morbidity and mortality worldwide. The aim of the study is to determine the relative contribution of the different pathogens to the overall disease burden. It will also determine the mechanisms of virulence of these pathogens that cause neonatal infections at Chukwuemeka Odumegwu Ojukwu University Teaching Hospital (COOUTH), Awka. Methods Biological samples were collected from 30 neonates admitted at the special care baby unit (SCBU) of COOUTH and cultured using selective media and nutrient agar. The isolates were identified using microbiological and biochemical tests. The antibiogram study was determined using Kirby-Bauer disc diffusion method on Mueller Hinton Agar. Several methods previously reported in literature were used for the characterization of the virulence factors. Results From the 30 blood samples collected, Pseudomonas spp. (19.7%), Escherichia coli (23%), Salmonella spp. (24.6%), and Staphylococcus aureus (32.8%) were isolated. Male to female ratio of study population was 1.5: 1. The isolates were 100 % resistant to ticarcillin, cephalothin, ceftazidime, and cefuroxime but appreciably susceptible to only levofloxacin (88.85%). They were moderately susceptible to ceftriaxone/sulbactam (39.05%) and azithromycin (26.46%). Common virulence factors identified among the isolates (up to 90 %) were hemolysin, biofilm formation, and acid resistance. Less common virulence factors were proteases (50 %), deoxyribonucleases (50 %), enterotoxins (63%), and lipopolysaccharide (70%). The virulence factors were found mostly among the S. aureus isolates. Conclusions Pseudomonas spp., Escherichia coli, Salmonella spp., and Staphylococcus aureus were implicated in neonatal infections in the center and most of them were resistant to conventional antibiotics. The organisms showed marked virulence and multidrug resistance properties. Levofloxacin, a fluoroquinolone, had superior activity on the isolates compared to other antibiotics used in the study.