The dihydroisocoumarin (3R,4R)-3,4-dihydro-4,6-dihydroxy-3-methyl-1-oxo-1H-isochromene-5-carboxylic acid (1) was isolated from Xylaria sp., a fungus associated with Piper aduncum (Piperaceae). Additionally, two known compounds, (R)-7-hydroxymellein (2) and (3R,4R)-4,7-dihydroxymellein (3), were isolated from Penicillium sp. associated to Alibertia macrophylla (Rubiaceae). The structures of all compounds were elucidated by analysis of spectroscopic data. The dihydrocoumarin derivatives 1–3 showed antifungal and acetylcholinesterase (AChE) inhibitory activities in vitro.