This study is based on the phenolic composition and the antioxidant, anticancer, antimicrobial, and antibiofilm activities of the edible mushroom Marasmius oreades from Turkey. The phenolic composition of an M. oreades ethanol extract was measured by using the Folin-Ciocalteu method, aluminium chloride colorimetry, and ultraperformance liquid chromatography. The antioxidant activity was evaluated on the basis of DPPH radical scavenging activity. The effect of the M. oreades ethanol extract was also screened in order to determine glutathione-S-transferase, glutathione peroxidase, catalase, and superoxide dismutase enzyme activities. The antimicrobial activity of the mushroom extract was evaluated by using well diffusion and was based on the minimum inhibitory concentration. In addition, the antibiofilm potential of M. oreades was analyzed against Gram-positive and -negative bacteria. Finally, the anticancer effects of the mushroom extract were tested on colon (HT-29) and breast (MCF-7 and MDA-MB-231) cancer cell lines by using the MTT assay. The results revealed that the total amount of phenolics in the ethanol extract of M. oreades was 10.990 ± 0.0007 mg gallic acid equivalent/100 g, and the total amount of flavonoids was 1.139 ± 0.0052 mg quercetin equivalent/100 g. The ultraperformance liquid chromatography results indicated that the M. oreades ethanol extract contained various phenolic compounds: catechin, ferulic, gallic acid, and vanillic acid. The M. oreades ethanol extract scavenged about 80% of DPPH free radicals. It did not show any effect on the glutathione-S-transferase, glutathione peroxidase, and catalase enzyme activities, but its maximal concentration (10 mg/mL) increased superoxide dismutase activity (8%). The ethanol extract of M. oreades showed a moderate anticancer effect on the HT-29, MCF-7, and MDA-MB-231 cell lines. Although the ethanolic extract of the mushroom did not show sufficient antibacterial activity, it presented a strong antibiofilm effect against all studied pathogenic strains at the tested concentrations.