Ganoderma resinaceum, a medicinal fungus with bioactive components present in it is not fully characterized for B-complex vitamins in the fruiting bodies. Identification and quantification of B-complex vitamins (thiamine [B1], riboflavin [B2], niacin [B3], and cobalamin [B12]) were conducted using reverse-phase high performance liquid chromatography (RP-HPLC) with a C18 column and UV detection at 280 nm from dry extracts of the fruiting bodies. Acid and enzymatic hydrolysis were used to extract these vitamins according to each specific analyte and the antioxidant capacity of the extracts was assessed against the DPPH radical in the concentration range of 12.5-200 µg/mL, based on ascorbic acid as a reference standard. Four vitamins were detected by well-resolved chromatographic elution peaks at 3.804 (B1), 4.824 (B3), 11.932 (B2), and 15.076 min (B12). B1 had the highest concentration (30.6 mg/L), followed by B2 (26.9 mg/L), B3 (24.8 mg/L), and B12 (1.8 mg/L). B1, B2, and B3 had DPPH scavenging activity in a concentration-dependent manner which was similar to the DPPH scavenging activity of ascorbic acid at high concentrations, whereas B12 had low DPPH scavenging activity at all concentrations tested. Collectively, results from the current study support both the identification and quantification of the four B-complex vitamins, and their nutritional value as a natural food source of B-complex vitamins for use in the development of functional foods and nutraceuticals.