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The rapid resistance developed by pathogenic microorganisms against the current antimicrobial pool represents a serious global public health problem, leading to the search for new antibiotic agents. The scorpion <i>Tityus stigmurus</i>, an abundant species in Northeastern Brazil, presents a rich arsenal of bioactive molecules in its venom, with high potential for biotechnological applications. However, venom cytotoxicity constitutes a barrier to the therapeutic application of its different components. The objective of this study was to produce <i>T. stigmurus</i>-venom-loaded cross-linked chitosan nanoparticles (Tsv/CN) at concentrations of 0.5% and 1.0% to improve their biological antimicrobial activity. Polymeric nanoparticles were formed with a homogeneous particle size and spherical shape. Experimental formulation parameters were verified in relation to mean size (<180 nm), zeta potential, polydispersity index and encapsulation efficiency (>78%). Tsv/CN 1.0% demonstrated an ability to increase the antimicrobial venom effect against <i>Staphylococcus aureus</i> bacteria, exhibiting an MIC value of 44.6 μg/mL. It also inhibited different yeast species of the <i>Candida</i> genus, and Tsv/CN 0.5% and 1.0% led to a greater inhibitory effect of <i>C. tropicalis</i> and <i>C. parapsilosis</i> strains, presenting MIC values between 22.2 and 5.5 µg/mL, respectively. These data demonstrate the biotechnological potential of these nanosystems to obtain a new therapeutic agent with potential antimicrobial activity.