Microbial leaching of a petroleum spent catalyst was carried out using mixed mesophilic iron and sulfur oxidizing acidophiles. Bench-scale batch stirred tank reactors with a working volume of 1 L were used in this study at 35 ℃. The pulp density considered for the study was 10% (w/v), while the particle size of the spent catalyst was varied by 45-106, 106-212 and >212 μm. The leaching percentage of Ni from the spent catalyst was found to be highest (97-98%) with varying particle size. However, the leaching yield for rest of the metals like Al, Fe, V and Mo was 70-74%, 66-85%, 33-43% and 22-45%, respectively. Influence of particle size was predominant on the recovery of all metals except Ni. Assessment of the generation of the bioleach residue after bioleaching showed a weight loss of 54-62% due to the dissolution of the metal values from the spent catalyst. The mineralogical study conducted by X-ray diffraction and scanning electron microscopy supports the dissolution of metals from the spent catalyst. Jarosite mineral phase was the dominant mineral phase in the bioleach residue due to the dissolution of the oxidic and sulfidic mineral phases present in the feed spent catalyst.