Background: This study evaluates the epidemiology and antimicrobial resistance profile of Gram-negative bacteria (GNB) and Gram-positive bacteria (GPB) isolated from clinical specimens in children admitted to Mofid Children's Hospital. Methods: This was a retrospective study of the patients' clinical specimens collected from January 2013 until the end of December 2018. All specimens were evaluated to determine the presence of infection-causing agents using a BACTEC 9120 blood culture. Isolation and identification of bacterial strains were performed using conventional biochemical tests. Antibiotic resistance was determined using Kirby-Bauer disk diffusion and broth microdilution methods. Results were interpreted according to CLSI and EUCAST. Results: A total of 1130 different pathogenic bacteria were detected from 14,690 different clinical specimens and the overall detection rate was 7.7% (1130/14,690). Among bacterial pathogen isolated from clinical specimens, 55% (n=622) were GNB and 45% (n=508) were GPB. The predominant GNB isolates were Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii, Escherichia coli, Enterobacter spp., Citrobacter spp., respectively. Among GPB, CoNS was the most frequent and Enterococcus spp. was found to have low levels of resistance to linezolid. In GNB, most A. baumannii and P. aeruginosa were ceftriaxone resistant. P. aeruginosa was found to have low levels of resistance to levofloxacin and ciprofloxacin. Conclusions: Our findings revealed that the resistance rate among GNB and GPB associated with different infections in children is very high. These results suggest a constant screening and follow-up programs for the detection of antibiotic resistance, and it also suggests to develop antimicrobial stewardship programs in Tehran, Iran.