Aim: The aim of the present study was to evaluate the pigment production by the fungus Fusarium chlamydosporum in Glucose Peptone Yeast Extract medium and further to assess its toxicity by in vitro methods. Materials and Methods: The coloured crude extract yielded 4 fractions when subjected to column chromatography, upon which Fraction 2 and 4 were subjected to characterization procedures by UV-Visible spectrophotometry, Fourier Transform Infrared Spectroscopy and Molecular weight determination by Liquid Chromatography Mass Spectroscopy (LCMS). The toxicity of the pigments was evaluated in vitro on L-929 Mouse Fibroblast cell lines by MTT Assay. Results: The fungus produced dark red pigment in Glucose Peptone Yeast Extract medium on extraction with ice cold chloroform. The results summarized Fraction 2 and 4 to be the polyketide derivatives, Rubrofusarin and 8-O-methyl fusarubin respectively wherein the IC50 values of rubrofusarin and 8-O-methyl fusarubin was 183.85 and 82.366 μg/ml respectively. Conclusion: The significance of the present study lies in the characterization of 2 novel polyketides, rubrofusarin and 8-O-methyl fusarubin from F. chlamydosporum and there have been no literature reviews on the production of these pigments from the fungus.