Ascomycete fungi are promising biofactories producing bioactive compounds exhibiting diverse biological activities. In an ongoing search for bioactive potential of microorganisms from different ecological niches, a promising Lasiodiplodia sp. strain B2 (MTCC 6000) was identified as a producer of exopolysaccharide (designated as Lasiosan). The exopolysaccharide was purified and structurally characterized as glucomannan having glucose and mannose residues (ratio 1:1) with average molecular mass of 29.3 kDa. Lasiosan demonstrated promising antimicrobial and anti-biofilm activities by inhibiting the growth of different Gram-negative, Gram-positive, drug-resistant bacteria and different Candida strains. The purified exopolysaccharide exhibited potential antioxidant activity in terms of good reducing power and scavenging of DPPH free radicals, superoxide anions and lipid peroxyl radicals. Further, the in vitro immune responses of Lasiosan were demonstrated in mouse RAW 264.7 macrophages. Lasiosan inhibited the LPS-stimulated reactive oxygen species (ROS) and nitric oxide (NO) generation in RAW 264.7 macrophages without affecting cell proliferation. Furthermore, Lasiosan significantly down-regulated the production of LPS stimulated proinflammatory mediators such as TNF-alpha and IL-6 from RAW 264.7 macrophages. This is the first report on Lasiosan exhibiting broad spectrum antimicrobial, anti-biofilm, antioxidant and immunomodulatory activities, which could be explored as a promising candidate for application in biotechnological and biomedical fields.