Melanogaster broomeanus Berk. is a type of truffle common in pine, oak, and hazelnut forests and which naturally spreads in the Black Sea, Mediterranean, South-West Anatolia in Turkey, is consumed as food and traded. Chemical investigation of M. broomenaus led to isolate of ten known compounds namely, brassicasterol (1), ergosterol (2), ergosterol peroxide (3), 5α, 6α-epoxy ergosta-7,22-diene-3β-ol (4), trametenolic acid (5), maleic acid (6), fumaric acid (7), mannitol (8), kojibiose (9) and trehalose (10). Assessments of the antioxidant, anticholinesterase, anti-urease, and cytotoxic properties of extracts and isolated compounds were carried out. Compound 5 showed the highest cytotoxic activity against H-1299 with IC50: 23.78±0.98 μg/mL, while compound 2 demonstrated most potent cytotoxicity against MCF-7 with IC50: 30.91±0.15 μg/ml. Also, compound 5 showed the highest antioxidant activity in all methods, particularly in DPPH and ABTS assays with IC50 values of 90.24±0.87, 75.41±0.10 µg/mL, respectively. Similarly, compound 5 exhibited highest activity with 38.84±1.22% and 44.58±0.96% inhibitions against AChE and BChE, respectively, while compound 7 were found to be most potent inhibitory against urease with value of 55.20±0.45% at 100 µg/mL concentration. These findings suggest that M. broomenaus, like numerous other edible mushrooms, is a steroid-rich truffle and a potential source of steroids.