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The prevalence of tuberculosis (TB) and human immunodeficiency virus (HIV) coinfection in Nigeria is currently around 19.1%. This indicates that the two diseases are still a burden on the nation"s health. The aim of this study was to evaluate the diagnostic microbiology capacity and the barriers in performing assay for TB and HIV at peripheral district-level hospital-based laboratories in Oyo State, Nigeria. Diagnostic microbiology capacity was estimated using a scale of 100-point where scores ≤ 49% were categorized as low, 50-79% fair and ≥80% good. Barriers to diagnosis were summarized in proportions. The diagnostic microbiology capacity revealed that 6 (35.3%) and 11 (64.7%) of the laboratories had "fair" and "low" capacity, respectively, to detect TB in cerebrospinal fluid/sputum. In testing for HIV, 3 (17.6%) of the laboratories had "fair capacity" and 14 (82.4%) had "low capacity" to detect CD 4 count and HIV antibodies in blood serum. The major diagnostic barriers in almost all (94.1%) the laboratories were lack of culture supplies and nonavailability of reagents/testing kits. There was no diagnostic microbiology service with good capacity to facilitate case detection of HIV and TB at the peripheral hospitals. Hence there is a need to improve the supply of reagents, culture stock and testing kits. This will facilitate the detection of TB and HIV cases in peripheral communities. IMPORTANCE This study provided a snapshot knowledge of testing capabilities and commodity availability at state laboratories. The findings should inform the action of stakeholders to improve diagnostic microbiology capacity, consequently enhancing diagnostic measures in detecting human immunodeficiency virus and Mycobacterium tuberculosis.