Bioremediation techniques are being developed as substitutes for physical–chemical methodologies that are expensive and not sustainable. For example, using the agricultural waste spent mushroom substrate (SMS) which contains valuable microbiota for soil bioremediation. In this work, SMSs of four cultivated fungal species, Pleurotus eryngii, Lentinula edodes, Pleurotus ostreatus, and Agaricus bisporus were evaluated for the bioremediation of soils contaminated by petroleum hydrocarbons (TPHs). The bioremediation test was carried out by mixing the four different SMSs with the TPH-contaminated soil in comparison with an unamended soil control to assess its natural attenuation. To determine the most efficient bioremediation strategy, hydrolase, dehydrogenase, and ligninolytic activities, ergosterol content, and percentage of TPHs degradation (total and by chains) were determined at the end of the assay at 40 days. The application of SMS significantly improved the degradation of TPHs with respect to the control. The most effective spent mushroom substrate to degrade TPHs was A. bisporus, followed by L. edodes and P. ostreatus. Similar results were obtained for the removal of aliphatic and aromatic hydrocarbons. The results showed the effectiveness of SMS to remove aliphatic and aromatic hydrocarbons from C10 to C35. This work demonstrates an alternative to valorizing an abundant agricultural waste as SMS to bioremediate contaminated soils.