Bioleaching of pre-treated spent refinery catalyst was performed using sulfur and iron oxidizing bacteria separately. Both the mixed cultures were grown at various concentration of either ferrous sulfate or elemental sulfur. Bioleaching process was conducted by varying the reaction time, effect of substrates and effect of catalyst amount. Under the most favourable transport conditions examined, (5g/L spent catalysts, 10g/L ferrous sulfate, reaction time 7 days) the mixed iron oxidizing bacteria were able to recover Ni, V, and Mo of 90, 80 and 54 % respectively. Higher catalyst and ferrous sulfate concentration showed decline result in recovery of Mo and V. Ni showed consistent results throughout the series of experiments (88 to 90% recovery). The mixed sulfur oxidizing bacteria were observed to be more effective than the iron oxidizing bacteria. The sulfur oxidizing cells were able to recover Ni, V and Mo of 88, 94 and 46 % respectively (spent catalysts 50g/L, elemental sulfur of 2% (w/v), reaction time 7 days). The oxidation of elemental sulfur to sulfate could play a vital role for extraction of the metals.