Objective: This study aimed to identify the bacteriological agentsof otitis media in Jordanian children and to assess the in vitro susceptibility of these isolates to commonly used antibiotics. Methods: Retrospective analysis of consecutive samples submitted for microbiological evaluation from outpatients children aged between 6 months and 15 years who were clinically diagnosed with otitis media and were treated at Princess Rahma Hospital in North Jordan between January 2005 and December 2009. Results: A total of 724 isolates were recovered from cultures obtained from 863 children patients giving an isolation rate of 83.8%. The male and female isolate ratio was (1.26:1.0). The most common bacterial species isolated were S. aureus (59.9%) followed by Streptococcus pneumoniae (22.4%), Pseudomonas (7.7%), E .coli (5.9%), Klebsiella spp. (3.1%) and Proteus spp. (0.9%). The most of S. aureus isolates were susceptible to vancomycin (94.0%) and gentamicin (87.3%). Streptococcus organisms were susceptible in highest percentage to amoxicillin-clavulanic acid (87.1%). Gramnegative isolates were highly susceptible to ciprofloxacin (83.5%) and gentamicin (79.8%). Among all isolates, vancomycin was themost effective antimicrobial agent with susceptibility rate of 83.9%, whereas cefixime showed the lowest susceptibility rate of 39.7%. Conclusions: S. aureus isolates were the most frequent bacteria isolated from otitis media and were highly sensitive to vancomycin and gentamicin, while gram-negative isolates were more sensitive tociprofloxacin and gentamicin.