Herein, comparative studies were conducted on different species of Pleurotus mushroom (Pleurotus osteratus, Pleurotus eryngii, Pleurotus sajor caju, Pleurotus florida, and Pleurotus roseus), extracted in two different solvent systems: dichloromethane (DCM)–ethanol (Et) with the ratio of 1 : 1 (v/v) and 50% (v/v) ethanol. Comparably, 50% (v/v) ethanolic crude extract had a considerably higher extraction yield and total phenolic, carbohydrate, and protein content. Owing to a high total phenolic yield, 50% (v/v) ethanolic crude extract had better ability to scavenge 2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid radical. Contrarily, DCM−Et crude extract showed strong cytotoxic potential against the human breast cancer cell line (MD-MBA-231) and murine skin cancer cell line (B16F10). High-performance thin layer chromatography based methods were developed and validated as per the International Council for Harmonisation Q2(R1) for the determination of ergosterol. Ergosterol content in DCM−Et crude extract correlates with its cytotoxic potential.