Aims: The aim of the study was to purify and characterize a bioactive compound from Aspergillus nidulans strain KZR-132 and its biological evaluation. Methods and Results: A bioactive extolite was purified from A. nidulans strain KZR-132, and its chemical structure was elucidated as 3-hydroxylbenzyl alcohol (3-HBA) based on H-1 and C-13 NMR, FT-IR and mass spectroscopic analysis. The antimicrobial efficacy of 3-HBA was established against Gram-positive, Gram-negative bacteria and different Candida strains. It also showed promising antibiofilm activity against various tested microbial strains. Reactive oxygen species induced by 3-HBA treatment on different Candida strains killed most of the cells and showed necrotic effect. It also exhibited dose-dependent antioxidant and anti-inflammatory activities. Conclusions: This bioactive extrolite produced by A. nidulans isolated from a niche habitat was demonstrated to possess significant biotechnological and pharmacological potential since it exhibited broad-spectrum antimicrobial and antibiofilm activities which are reported for the first time. Significance and Impact of the Study: The overall study demonstrates that 3-HBA produced by A. nidulans KZR-132 is a promising bioactive metabolite and possibly can function as a pharmacologically suitable broad-spectrum antimicrobial drug candidate against various dreaded human-related bacterial and fungal pathogens.