Adaptation of Penicillium simplicissimum with different heavy metals present in a spent hydrocracking catalyst, as well as one-step, two-step, and spent medium bioleaching of the spent catalyst by the adapted fungus, was examined in batch cultures. Adaptation experiments with the single metal ions Ni, Mo, Fe, and W showed that the fungus could tolerate up to 1500 mg/L Ni, 8000 mg/L Mo, 3000 mg/L Fe, and 8000 mg/L W. In the presence of multi-metals, the fungus was able to tolerate up to 300 mg/L Ni, 200 mg/L Mo, 150 mg/L Fe and 2500 mg/L W. A total of 3% (w/v) spent catalyst generally gave the maximum extraction yields in the two-step bioleaching process (100% of W, 100% of Fe, 92.7% of Mo, 66.43% of Ni, and 25% of Al). The main lixiviant in the bioleaching was shown to be gluconic acid. The red pigment produced by the fungus could also possibly act as an agent in Al leaching.