Purpose: To determine the association between isolates in the middle ear (ME) and nasopharynx of patients with chronic otitis media in Ilorin, north-central Nigeria. Methods: An ethically approved case control study was carried out in the Ear, Nose, and Throat clinic amongst consenting cases using normal subjects as controls. A microbiology investigation form giving the results for otoscopy, aspirate and swabs was filled out for both the ME and nasopharynx. The experimental procedure was carried out and bacteria were identified according to colony characteristics, morphological appearance, Gram-staining, and standard biochemical testing. Data obtained were analysed with SPSS version 16.0 and Epi Info 3.5.1 using the mean, standard deviation and chi-square results. Results: A total of 140 cases and 70 controls, were recruited. The Gram stain reaction of the ME aspirates were positive in 28.6% and negative in 71.4% of cases. Nasopharyngeal swabs revealed 64.3% Gram positive and 35.7% negative organisms. Overall, there was no relationship between the ME and nasopharyngeal isolates amongst cases, with a P value of 0.000. However, there was a relationship amongst the isolate from the nasopharynx of cases and controls, with the exception of Klebsiella pneumoniae, at P < 0.009. Conclusion: There was no relationship amongst the bacterial isolate from the ME and nasopharyngeal specimen of patients with otitis media.