The occurrence of several weeds in crops of high economic value directly affects grain yield. This scenario led to the mass investigation of chemical products that circumvent these adversities and provide control potential. Nonetheless, the excessive application of chemical herbicides has generated significant concerns about the environment. Accordingly, the adoption of alternative practices, such as the application of microbial metabolites, emerges as strategic control actions, having a sustainability bias and allowing the reduction of risks of human and animal contamination. Appropriately, this study proposed to conduct a microbial prospection of microorganisms capable of producing secondary metabolites to inhibit growth and generate phytotoxicity in weeds. Furthermore, to increase the herbicidal activity, different strategies were evaluated involving microbial co-cultivation and ultrasound-assisted extraction after fermentation. Accordingly, 63 microorganisms were isolated from weeds with disease symptoms and submitted separately to submerged fermentation. Initially, the bioherbicidal activity was evaluated in Cucumis sativus plants, and the most promising were applied in Amaranthus hybridus and Echinochloa crusgalli. Treatments with fermented broth obtained from co-cultivation and ultrasound-assisted extraction after the fermentation process indicated an inhibition of plant development. The most promising strains were A14.2 and B22.2 (Nigrospora sphaerica), B14 (Bacillus velezensis), and γ (Aspergillus flavus).
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