Edible mushrooms have been considered an excellent source of nutraceuticals with high potential to prevent diseases such as cancer. Recently the anti-cancer potential of a small RNA (sRNA) fraction isolated from C. cibarius was described. To better understand its molecular nature, a study was carried out regarding the anti-cancer potential of sRNAs isolated from different mushroom species. Apart from the isolation of RNA-rich fractions by anion exchange chromatography, miRNA’s extraction was also carried out using a commercial kit. The anti-proliferative activity of the different samples was tested in normal and cancer cell lines. Our data demonstrated differences regarding sRNA anti-cancer activity in different mushroom species, and this suggests that the sRNA fractions with anti-cancer potential are enriched in a specific sequence. These results reaffirm mushrooms as a source of bioactive compounds. In particular, the RNA-enriched isolated fractions demonstrated important anticancer potential.