The present study is focused on the assessment of the antimicrobial activity of cerumen and antibiotics against bacteria isolated from ear pus samples. Thus, a total of 50 ear pus samples were collected from infected patients using sterile swabs and were screened using pure culture techniques. Total of 04 different bacterial isolates were identified while, the prevalence data revealed that Pseudomonas spp., were dominant (58%, n = 29) among isolated bacteria followed by Staphylococcus spp., (22%, n = 11), Escherichia coli (14%, n = 7) and Proteus spp., (6%, n = 3). Further, bioassay revealed that Pseudomonas spp., and Staphylococcus spp., were most sensitive to Clindamycin (94.73%) while displayed resistant to Ciprofloxacin and Ampicillin. Similarly, E. coli and Proteus spp., were most sensitive to Ciprofloxacin (92.8-95.21%) as compared to the other antibiotics. Moreover, antibacterial activity of cerumen was also assessed against test organisms and its maximum activity was observed against Pseudomonas spp., (90% equivalent to Clindamycin potency) and Staphylococcus spp., (60% equivalent to Amoxicillin potency) while least effective against E. coli (36%) and Proteus spp., (22%). Thus, it was concluded that the antibacterial activity of cerumen might be due to the presence of potential chemicals i.e. flavonoids and terpenoids.