Sixty-five Gram-negative bacterial isolates were collected from different samples (blood, urine, stool, cerebrospinal fluid (CSF) and wound swabs) from Mansoura University Children's Hospital, and identified as Klebsiella pneumoniae  (n= 10), Escherichia coli (n= 8), Enterobacter aerogenes (n= 7), Enterobacter cloacae  (n= 6), Serratia fonticola (n= 6), Citrobacter freundii complex (n= 5), Kluyvera ascorbata (n= 4), Acinetobacter baumannii/haemolyticus (n= 3), Pseudomonas aeruginosa (n= 2), Pseudomonas fluorescens⁄putida (n= 2), Vibrio alginolyticus (n= 2), Vibrio damsela (n= 1), Cedecea lapagei (n= 1), Cedecea species 3 (n= 1), Proteus mirabilis (n= 1), Providencia stuartii (n= 1), Serratia marcescens (n= 1), Serratia plymuthica (n= 1), Serratia liquefaciens (n= 1), Stenotrophomonas maltophilia (n= 1) and Acinetobacter lwoffii (n= 1). Thirty-one antimicrobial agents were tested against the isolated bacteria. Gatifloxacin, imipenem and meropenem were found to be the most effective agents against the isolated bacteria, while ampicillin, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, gentamicin and tobramycin were found to be of low effect against the isolated bacteria.