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The microbial characterization of two spent mushroom substrate (SMS) from Pleurotus osteratus (Jacq. ex Fr.) P. Kumm and Pleurotus tuber-regium (Rumph. ex Fr.) Singer were investigated. The microbiological properties of the spent mushroom substrate were determined using standard methods. Quantitative microbiological analysis revealed that P. osteratus spent mushroom substrate (POSMS) contained 7.9 × 105 and 1.2 × 103 cfu g-1 of total heterotrophic bacteria and total fungi count, respectively, while P. tuber-regium spent mushroom substrate (PTSMS) contained 1.38 × 106 and 9.0 × 102 cfu g-1 of total heterotrophic bacteria count and total fungi count respectively. The fungal species from PTSMS included Aspergillus and Cladosporum species, while Aspergillus and Penicillium species were found in POSMS. The bacterial species encountered from PTSMS included Bacillus, Acinetobacter, Alcaligenes, Actinobacter and Pseudomonas species while Bacillus, Actinobacteria, Aeromonas, Lactobacillus and Aerococcus species were encountered from POSMS. Therefore, based on the findings from this study it can be concluded that spent mushroom substrates contain microorganisms that can be utilized both in bioremediation of oil polluted soils as they contain important hydrocarbon utilizing microorganisms such as Penicillium, Aspergillus and Bacillus species and also as sources of plant growth promoting rhizobacteria (PGPR) such as Pseudomonas and Bacillus species which can induce resistance on plants. However, further studies are recommended to confirm these findings further.