Background: Bacterial infections of ear canal are widely prevalent. Objective: This study was planned to, diagnose cases of otitis externa then make isolation and identification of the most prevalent bacterial pathogens causing this infection. Also, to detect the phenotypic antimicrobial susceptibility testing to select the effective antimicrobials. Subjects and methods: A prospective clinical study was conducted on 100 cases with acute otitis externa. Patients’ socio-demographic features and clinical history were recorded. Identification of the bacterial pathogens and surveying of antimicrobial resistance and resistance genes were conducted. The efficacy and minimum inhibitory concentrations (MIC) of cerumen, human serum, and silver nanoparticles against the pathogens were detected. Results: A total of 100 acute otitis externa patients were investigated, 25% had tonsillectomy. Most of the cases showed discomfort, itching, otalgia, and edema of the ear canal. Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were highly prevalent with rates of 30%, 20%, and 15% respectively. They were highly resistant with multiple antimicrobial resistance indices of 0.75, 0.7, and 0.95 respectively. S.aureus contained high rates of mecA1, A2, mecA1, mecC, and BlaZ gene. Most of E. coli harbored class 1 integron, CTX-M, and CTX-M-1. Most of the macrolide-lincosamide–streptogramin B methylases were present in S.aureus and E.coli. P.aeruginosa contained class 1 integron, while low rates of CTX-M, msrE, mphE. Silver nanoparticles MIC was 650 μg/mL against E.coli and P.aeruginosa with inhibition zones of 30 mm and 20 mm, respectively. The cerumen MIC was 1200 μg/mL against S.aureus and E.coli with inhibition zones of 7 mm and 9 mm respectively. Human serum was effective against E.coli with a MIC of 200 μL/mL and the inhibition zone was 13 mm. Conclusions: Bacterial pathogens of otitis externa exhibited high antimicrobial resistance rates. Cerumen, human serum, and silver nanoparticles are promising in controlling these pathogens.