Increase in productivity is directly related with increase pesticide consumption. So there is a strong desire to use "greener" chemistry to produce more toxicologically and environmentally benign pesticides and natural products. In this present study, a comparison was made to evaluate the phytotoxicity potential of first cleanup fractions obtained from sequentially extracted solvent (ethyl-acetate, methanol) extracts of Parthenium hysterophorus L. (aerial parts) in vitro through bench-top seed germination assay (Vigna radiata L.). One-way analysis of variance (ANOVA) followed by Duncan's multiple range test (DMRT) were done for statistical analysis of the data. The study reveals that germination, growth and vigour was significantly (P<0.05) reduced by ethyl-acetate and methanol fractions. A significant changes in soluble and insoluble sugar content, protein, amino acid content and cellular metabolic activity was aslo observed by 1F3, 1F4, 1F6, and 1F9 fractions. The present study concluded that phytotoxicity of four fractions (1F3, 1F4, 1F6, and 1F9) from ethylacetate and methanolic crude extracts of Parthenium hysterophorus could be exploited as potential bioherbicide for future weed management programme and the development of bioherbicide for commercial use.