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Production of a bioherbicide for biological control of weeds requires a series of steps, from selection of a suitable microbial strain to final formulation. Thus, this study aimed to select fungi for production of secondary metabolites with herbicidal activity using biological resources of the Brazilian Pampa biome. Phytopathogenic fungi were isolated from infected tissues of weeds in the Pampa biome. A liquid synthetic culture medium was used for production of metabolites. The phytotoxicity of fungal metabolites was assessed via biological tests using the plant Cucumis sativus L., and the most promising strain was identified by molecular analysis. Thirty-nine fungi were isolated, and 28 presented some phytotoxic symptoms against the target plant. Fungus VP51 belonging to the genus Diaporthe showed the most pronounced herbicidal activity. The Brazilian Pampa biome is a potential resource for the development of new and sustainable chemical compounds for modern agriculture.