Omphalitis is a hatchery-borne disease responsible for first-week chick mortality. The present study aimed to isolate and investigate the antimicrobial resistance profile of bacteria causing omphalitis in broiler farms and then investigate the antibacterial properties of chitosan nanoparticles against isolated bacteria. In this study, 100 living diseased chicks were collected from 10 broiler chicken farms. Overall, 86 bacterial isolates were successfully recovered and then identified as 44 E. coli, 21 Salmonella, 13 Pseudomonas, and 8 Staphylococcus. E. coli isolates were serotyped as EPEC (O₂H₆, O₁₂₁H₇ & O₅₅H₇), ETEC (O₁₂₈H₂ & O78), EHEC (O₂₆H₁₁ & O₉₁H₂₁) and EIEC (O₁₅₉). Meanwhile, Salmonella isolates were serotyped as S. enteritidis, S. larochelle, S. typhimurium, S. kentucky and S. takoradi. Pseudomonas and Staphylococcus isolates were serologically confirmed as 13 Pseudomonas aeruginosa and 6 Staphylococcus aureus. The antimicrobial resistance profile of tested isolates against sixteen commercial antibiotics showed a high multiple antibiotic resistance index. Agar well diffusion test indicated that chitosan nanoparticles were in vitro effective against the most resistant bacteria isolates. The minimum inhibitory concentration of chitosan nanoparticles for E. coli (O78), S. kentucky, P. aeruginosa and S. aureus were 1.25, 2.5, 2.5 and 10 mg. ml^-1, the present results indicated that chitosan nanoparticles are effective in vitro against the bacterial pathogens inducing omphalitis in broilers chicks.