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Background: Fungal endophytes produce a variety of secondary metabolites which can be explored for herbicidal activities. Partheniumis a very aggressive and fast-spreading weed in Pakistan. The endophytes of Partheniummade it superior over other weeds. Those endophytes can be isolated, identified, and incorporated in sustainable agriculture to biologically control other severe weeds. Main body: The present study was conducted to isolate and identify the endophytic fungi from the roots of Parthenium hysterophorus(Family: Asteraceae) and evaluate their cultural filtrate against the growth of three selected weed species, i.e., Chenopodium album, Avena fatua, and Convolvulus arvensis. Moreover, pot experiments were conducted to evaluate the effects of cultural filtrates of these endophytic fungi against the germination, growth, and other physiological parameters (cellular contents, respiration, and cellular leakage) of the abovementioned three weed species. Three endophytic fungi, i.e., Alternaria, Aspergillus, and Drechsleraspp., were isolated from the roots of P. hysterophorus. Among these endophytic fungi, cultural (hyphal) filtrate of Alternariaspp. exhibited the strongest phytotoxic effect, followed by Drechsleraspp. and Aspergillusspp. against the selected weeds. Moreover, all the three endophytic fungi showed significant reduction in the germination as well as other physiological parameters of the weed species. Conclusion: It is concluded that the utilization of endophytic fungi could be a non-chemical, effective, ecofriendly, and low-cost biological control method that can be used for the biomanagement of fast-spreading and aggressive weeds.