The present study was focused to study the production of secondary metabolite by the fungus, F. chlamydosporum on a non-defined medium with less concentration of nitrogen; the organic nitrogen source being peptone and beef extract. In this context, we have been successful in extracting a polyketide pigment from the fungus by using the homogenization technique. The pigments thus extracted were subjected to various purification techniques via thin layer chromatography, column chromatography, UV-visible spectrophotometry, fourier-transform infrared spectroscopy and finally molecular determination by liquid chromatography coupled to tandem quadrupole mass spectrometry (LC-MS-MS). The polyketide red pigment was finally characterized and identified to be fusarubin following which its cytotoxicity was evaluated in vitro by using normal lung fibroblast cell lines (MRC-5). In the verge of researchers identifying novel compounds for various applications, the production of fusarubin by the fungus can be a major breakthrough as fusarubin has been investigated to exhibit many pharmacological activities. Though fusarubin is reported to be produced by other Fusarium species, this is the foremost study on the production of fusarubin by F. chlamydosporum; the composition of the culture medium is also unique. The production of this polyketide probably correlates in the pathogenesis of F. chlamydosporum as studies comment on this fungus as an opportunistic pathogen.