The physicochemical characteristics of oyster mushrooms (Pleurotus ostreatus) cultivated using liquid spawn (MLS) were compared with those of commercial mushrooms cultivated using solid spawn. The color intensity of the two types of mushrooms showed no remarkable difference. The hardness of the MLS-cultivated mushrooms was significantly higher, but their moisture content (86.80%) was significantly lower than that of the commercial mushrooms. Mineral contents in MLS-cultivated mushrooms (421.17 mg/100 g) were significantly higher than those in the commercial mushrooms (333.26–362.78 mg/100 g); in particular, the potassium (K) content was the most abundant in the former. The amino acid content in the MLS-cultivated mushrooms (4,695.22 mg/100 g) was about 1.4–2.0 times that in the commercial mushrooms. The essential amino acid contents and sum of aspartic acid and glutamic acid were higher in the MLS-cultivated mushrooms than in the commercial mushrooms. The β-glucan content in the MLS-cultivated mushrooms was 1.1–2.3 times higher than that in the commercial mushrooms. The total phenol and flavonoid contents and the DPPH and ABTS radical-scavenging activities of the MLS-cultivated mushrooms were significantly higher than those of the commercial mushrooms; however, the reducing power showed an opposite trend. Therefore, MLS-cultivated mushrooms contained higher amounts of valuable components and higher antioxidant activities than commercial mushrooms.