A novel antifungal antibiotic designated fumifungin (1) was isolated from the culture filtrate of Aspergillus fumigatus Fresenius 1863 (strain Y-83,0405) isolated from a Himalayan soil sample. This paper reports the isolation, purification, structural elucidation, and antifungal activity of fumifungin. The organism was cultured in seed medium and fermented in production medium. Fumifungin was purified using Diaion HP-20 resin column chromatography and reverse-phase (octadecylsilyl) chromatography. Structural analysis via fast atom bombardment mass spectrometry (FAB-MS), high-resolution electron impact mass spectrometry (HREI-MS), nuclear magnetic resonance (NMR) spectroscopy, and elemental analysis established fumifungin as 2-amino-4-acetoxy-3,5,14-trihydroxy-Δ6-eicosenoic acid. Antifungal activity testing showed varying minimum inhibitory concentration (MIC) values against selected fungi including Candida albicans, Saccharomyces cerevisiae, Aspergillus niger, Penicillium digitatum, Trichophyton mentagrophytes, and others.