BackgroundUse of phytopathogenic fungi for the biocontrol of weeds represents a promising path in the search for new management alternatives that allow reducing negative effects on the environment and the generation of biotypes having resistance to herbicides. The first step in developing weed biological control programs is to determine the plants and their natural enemies with the highest affinity and potential to achieve effective biocontrol. The objective of the present study was to evaluate the phytopathogenic potential of fungal isolates on four economically important weeds including: Rumex crispus L., Digitaria horizontalis Willd, Persicaria nepalensis (Meisn.) Miyabe, and Thunbergia alata Bojer ex Sims, as a possible biocontrol agent.ResultsMorphological and molecular identification of nine phytopathogenic isolates of weeds was achieved, according to the sequencing of the ITS, β-Tub2, and TEF1-α regions. Pathogenicity of the following species on original hosts was confirmed: Colletotrichum cigarro, Epicoccum draconis, and Didymella rumicicola on R. crispus; Bipolaris sp., on D. horizontalis; Bipolaris zeicola, Phialemoniopsis curvata, and Stemphylium beticola on P. nepalensis and, Alternaria thunbergiae and Nigrospora sphaerica on T. alata. These could be, fairly, considered the first worldwide reports of such interactions, except for A. thunbergiae and Bipolaris sp. The most virulent interactions according to the AUDPC value corresponded to (R. crispus × D. rumicicola), (D. horizontalis × Bipolaris sp.), (P. nepalensis × S. beticola) and (T. alata × A. thunbergiae), with an incidence of 100%.ConclusionThese strains were proposed for future research as potential biocontrol agents, which represented a great resource for the possible generation of new bio-herbicides.