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Abstract Mandacaru (Cereus jamacaru DC.), is a cactaceous symbol of caatinga vegetation at Brazilian Northeast region, however, there are no much studies about biochemical properties of this species. Here, the pioneering study brings very relevant data to highlight the importance of research with endemic plants of the caatinga. Afterward, the presence of enzymes such as peroxidase, protease, chitinase, β-1,3-glucanase, and serine (trypsin) and cysteine (papain) protease inhibitors were evaluated. The peroxidase activity was higher in roots than other tissues. The β-1,3-glucanase and proteolytic activity were prominent in stem and roots. The chitinase activity and protease inhibitor for both classes analyzed were detected in the stem and fruit peel. Antifungal activity against Colletotrichum gloeosporioides showed the root extract has a promising inhibitory activity on this economical important phytopathogenic fungus. After the contact of the hyphae with root extract increase in membrane permeability, based on Propidium Iodide (PI) uptake, and production of reactive oxygen species (ROS) were detected, compared to negative control. In addition, Scanning Electron Microscopy (SEM) analysis showed morphological damage on hyphae structure indicating that the treatment debilitates either cell membrane or cell wall leading to the cell death C. gloeosporioides. Graphical abstract Image 1 Highlights • C. jamacaru is a source of antimicrobial proteins with biotechnological potential. • C. jamacaru has proteo-, chitinolytic, protease inhibitor, and peroxidase activity. • C. jamacaru roots extract inhibit fungal growth. • Root proteins have antifungal activity by inducing membrane pore formation. • SEM microscopy showed loss of internal content in treated fungi. [ABSTRACT FROM AUTHOR]