From an antimicrobial gram-positive actinomycete strain of Saccharomonospora azurea (MTCC11714) isolated from high altitude soil of Kargil (J&K, India), a new isocoumarin saccharonol B (2) along with two known compounds viz. saccharonol A (1) and piericidin A3 (3) was isolated and characterized. The structure of the new compound was established based on extensive 2D-NMR data. Saccharonol B (2) exhibited mild antimicrobial activity against a standard panel of microorganisms Staphylococcus aureus ATCC 29213, Candida albicans ATCC 90028, and Aspergillus fumigatus MTCC 1811 with MIC values in the range of 128–248 lg/mL. Saccharonol B (2) and piericidin A3 (3) showed selective cytotoxic activity against human pancreatic carcinoma cell line (MIAPaCa-2) with IC50 values of 9 and 8 lM, respectively. Mechanistic studies indicated that saccharonol B (2) arrests S-phase of the cell cycle and causes dose-dependent loss of mitochondrial potential in MIAPaCa-2 cells.