The aim of this study was to investigate the prevalence and antimicrobial susceptibility of bacterial strains isolated from tracheal specimens obtained from pediatric patients admitted to a major children hospital in Tehran, in 2007. Tracheal specimens were cultured on the appropriate bacteriological media. Bacterial isolates were identified by standard biochemical and serological tests. Antimicrobial susceptibility testing was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Pseudomonas spp. was identified as the most prevalent bacterial isolate (32%) followed by Staphylococcus aureus (27.6%). Thirty strains (16%) were identified as Klebsiella spp., 18 (9.6%) as Enterobacter spp. and the rest belonged to coagolase negative Staphylococci, Streptococcus viridans, Acinetobacter spp., Escherichia coli and Neisseria spp. All Pseudomonas spp. were resistant to ampicllin, kanamycin and ceftizoxime. Staphylococcus and Klebsiella spp. showed high degree of resistance to 40% of examined antibiotics.