The present study was designed to evaluate the scaling process used to obtain fungi and enzymes fromTrichoderma koningiopsis to produce bioherbicides with low levels of genotoxicity using a stirred-tank bioreactor. We measured fungal biomass and activities of enzymes (amylase, cellulase, lipase and peroxidase). Fermentative products were applied to weeds (Bidens pilosa andEuphorbia heterophylla), and the bioherbicide effect was evaluated. We found greater elevations of peroxidase and lipase activity in the scaled-up models than in those at small scale (0.15 L), even with less biomass, suggesting that the scaling was cost-beneficial in terms of enzymatic yield. Bioherbicide activity tests showed up to 100–80% phytotoxic effects on weeds that were resistant to synthetic herbicide. A genotoxicity study of the fermented extract performed inAllium cepa meristem cells showed low genotoxicity and no DNA damage. Our findings point to new perspectives regarding bioherbicides derived from fungal fermentation and enzyme yield technology, aimed at sustainable and ecofriendly agriculture.