Weed control by herbicide is issued for environmental problems and the emergence of resistance herbicide; thus, researchers are looking for alternative methods including bioherbicide. Hence, this study aims to isolate the pathogenic fungi that associated with parthenium weed (a significant noxious weed as a threat to agriculture) and then extract the fungal mycelia for application on seed germination inhibition to be potential as bioherbicide to control parthenium. In this study, the fungus of Aspergillus sp. and Valsa mali were isolated from parthenium leaf and their mycelium were extracted to isolate secondary metabolites using ethyl acetate solvent from the culture of potato dextrose broth (PDB) and malt extract broth (MEB) mediums. In vitro, both fungal metabolites were applied on seeds in plate assay experiment. Original and diluted culture filtrates of Aspergillus sp. inhibited the seed germination by 51% and 20% in PDB and 48% and 39% in MEB respectively, over control. Similarly, Original and diluted culture filtrates of Valsa mali significantly suppressed the seed germination by 52% and 24% in PDB and 62% and 33% in MEB respectively, over control. Therefore, it indicated that fermented culture mycelia metabolites from these fungi able to inhibit seed germination efficiently and can be potentially used as bioherbicide to control parthenium weed.