Otitis media (OM) is a worldwide major health and economic issue leading to hearing loss, especially in developing countries due to high antibiotic resistance among the causative pathogens. This study aims to isolate, purify and determine the prevalence of MDR microorganisms causing OM. This is in addition to estimate in vitro effectiveness of irradiated and un-irradiated aqueous garlic extract (AGE). Finally, the present study aims to evaluate the enhancement of CIP, TOB, and NYS, commonly used in OM treatment, with IAGE against identified MDR bacteria and fungi. In the current investigation, the in vitro data revealed that OM is more prevalent in middle aged adults than in children. OM were predominated by bacterial isolates (59.0%), followed by fungal isolates (41.0%), including Pseudomonas aeruginosa (5 isolates), Proteus mirabilis (3 isolates), in addition to Klebsiella pneumoniae, Alcaligenes faecalis, Enterococcus faecalis, Bacillus cereus, Penicillium chrysogenum, Aspergillus flavus, and Aspergillus niger (1 isolate). Irradiated AGE (IAGE) showed nearly the same antimicrobial activity as the un-irradiated AGE. The inhibition activity enhancement was more statistically significant (P< 0.001) on combination of IAGE with CIP\NYS than with TOB (P< 0.05) and (P< 0.001) compared with each alone. Noticeable morphological changes in P. aeruginosa and A. flavus were observed by TEM images after the combination of IAGE with CIP/NYS. This study is one of primarily attempts to evaluate the IAGE to be used in future as a natural, safe, sterile, low cost, and available whole additive in therapeutic protocols to overcome MDR problem