Colletotrichum musae is a major pathogen in banana fruit which causes heavy losses at the postharvest stage. As a viable alternative to harmful chemical usage and physical control measures, the potential of using bacterial antagonist against C. musae was evaluated in this study. Out of a total of 92 bacterial isolates derived from dilution plates of aqueous extracts of spent mushroom substrate (SMS), 43 isolates were initially selected for screening for antagonistic activity against the C. musae isolate CMK01. Screening of the selected bacterial isolates revealed that 39 of the tested isolates significantly inhibited fungal growth in vitro compared to the control (P <0.05). The diffusible substances secreted by the five selected isolates showed 100% inhibition of colony growth, while (13-58%) inhibition was observed with volatile compounds. Light microscopic observations revealed deformation and blackening of C. musae hyphal tips in the colony margins facing the antagonists. Four of the selected five bacterial isolates were identified with 100% precision as Pseudomonas aeruginosa based on partial 16S rDNA sequences. These promising antagonists have the potential to be developed as effective biocontrol agents against C. musae.