To further knowledge of the biological activity of native neotropical fungal species, this study aimed to determine the chemical composition and microbiological activity of Hornodermoporus martius. Ethanol, hexane, diethyl ether, and ethyl acetate fractions and the water residue were analyzed and resulted in a total phenolic compound content between 13 and 63 mg of gallic acid equivalents per gram of crude extract. The total antioxidants ranged between 3 and 19 mg of ascorbic acid equivalents per gram of crude extract, and the percentage of antioxidant activity was determined to be between 6 and 25%. A preliminary profile of compounds is provided for the first time for the species; the results from the nonpolar fraction showcased the presence of saturated and unsaturated acids, fatty alcohol, sterols, and cis-vaccenic acid. Our findings also revealed antimicrobial properties from compounds within the hexane and diethyl ether fractions at concentrations of 1 mg mL-1, which inhibited the growth of certain gram-positive and gram-negative bacteria. For the first time in academic literature, our work analyzed and documented the chemical characteristics and microbial properties of H. martius, suggesting potential for medicinal applications.