Scleroderma is a genus of Gasteromycetes and Basidiomycota mushrooms in tropical and subtropical regions. This study aimed to evaluate the ethanolic extract of the mushroom Scleroderma citrinum regarding phytochemical prospecting, vitamin, phenolic, and total flavonoid content, and antioxidant (FRAP and DPPH) and acetylcholinesterase (AChE) inhibition activities. Fruiting bodies of S. citrinum were collected and the ethanolic extract was produced by maceration. Phytochemical prospecting was performed for several phytochemical groups using colorimetric means; the content of vitamins A, B, C, D, and E was obtained qualitatively by colorimetric methods, the content of phenolic and flavonoids by the colorimetric method and quantification by spectrophotometry. The spectrophotometric method performed the antioxidant activity in reducing FRAP and DPPH radicals by spectrophotometry and the acetylcholinesterase inhibition activity. Seventeen positive phytochemical groups were observed, the qualitative presence of vitamins of the A, B, and D complexes, 195.03 mg GAE g-1 of total phenolics, 93.10 mg QE g-1 of total flavonoids, FRAP reduction of 3.941 µM TE g-1, DPPH reduction of 127.78 µg mL-1 and AChE inhibition of 55.6%. The extract of the mushroom Scleroderma citrinum proved to be rich in phytocompounds, vitamins, and important biological antioxidant and acetylcholinesterase inhibition effects.