Mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest owing to their diverse roles in biological systems and are far from being completely understood. Even though, cellular cargo release mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study we investigate this aspect in detail using Raman micro-spectroscopic imaging. We provide considerable evidence to suggest that the release of antibiotic penicillin by filamentous fungus Penicillium chrysogenuminvolves EVs. Morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs are also revealed in this study.