Mushroom cultivation generates enormous amounts of post-cultivation substrate at the end of each production cycle, which is known as spent mushroom substrate (SMS). Once known as a waste product of mushroom production, SMS is actually a valuable co-product of mushroom production. There are countless possibilities for using this material and, considering the large volumes produced, it is interesting to continue looking for new alternatives for its use in different human activities. In this work, we evaluated the potential for biosurfactant production in SMS from different species of oyster mushrooms, in addition to evaluating the potential for oil degradation in the presence of SMS from Pleurotus ostreatus and Lentinula edodes. Even with the use of dry SMS, a decrease in several hydrocarbons was observed after 3 months of oil treatment. Furthermore, Pleurotus species showed high emulsification rates. Future studies will be necessary to characterize the molecules responsible for the bioemulsification activity, since both biosurfactants and bioemulsifiers can act in a similar way.