Lentinus arcularius (=Polyporus arcularius) is a medicinal polypore mushroom recorded in Vietnam based on morphological observation. In this study, three L. arcularius isolates were identified using molecular and morphological methods. Furthermore, the antimicrobial and antioxidant activities of their intracellular and/or extracellular metabolites were investigated. The ethyl acetate and n-butanol fractions were the most active extracts against Gram-positive and Gram-negative bacteria (Escherichia coli VTCC 12014, Pseudomonas aeruginosa VTCC 12273, and Staphylococcus aureus VTCC 10658) and fungi (Candida albicans VTCC 20568, Saccharomyces cerevisiae VTCC 20146, and Aspergillus niger VTCC 30001). These fractions also showed the highest antioxidant activity by α,α-diphenyl-β-picryl-hydrazyl as a free-radical agent or the highest percentage at 75-100% inhibition. These results suggest that L. arcularius might be a potential medicinal mushroom with antimicrobial and antioxidant effects.