Microbial preparation of herbicide is defined as bioherbicide that can control the weed. In this approach, indigenous plant pathogens isolated from weeds are cultured to produce the large numbers of infective propagules which are applied at a rate that will cause high levels of infection leading to suppression of the target weed. During the present investigation, cell free culture filtrate (CFCF) of Alternaria alternata was evaluated for its phytotoxicity against a noxious weed Lantana camara. The results of cut shoot, seedling and detached leaf bioassays revealed the presence of a toxic metabolite in the CFCF and a significant reduction in chlorophyll and protein content were also noticed. Phytotoxic moiety was further purified and characterized by using solvent partition, thin layer chromatography (TLC), FTIR and 1H NMR analysis. The acetone extract induced maximum phytotoxic damage at a concentration of 100 μg/ml and TLC purified fraction also exhibited herbicidal potential. The toxic compound was identified as tenuazonic acid upon comparison with FTIR and 1H NMR spectra. This is the first evidence that confirmed the herbicidal potential of a biorational, tenuazonic acid was produced by submerged fermentation of A. alternata.