From the ethyl acetate extract of the culture broth of Talaromyces verruculosus, a rhizosphere fungus of Stellera chamaejasme L., (−)-8-hydroxy-3-(4-hydroxypentyl)-3,4-dihydroisocoumarin (1) and (E)-3-(2,5-dioxo-3-(propan-2-ylidene)pyrrolidin-1-yl)acrylic acid (2) were isolated and evaluated for their antimicrobial activities. Their structures were elucidated by UV, IR, MS, 1H-NMR, 13C-NMR and 2D NMR spectra. Compound 1 exhibited the significant activities in vitro against two strains of bacteria and four strains of fungi. Compound 2 gave slight activities on the fungi at 100 µg mL−1, but no activities on the bacteria. Compound 1 should be considered as a new lead or model compound to develop new isocoumarin antimicrobial agents.