Antimicrobial peptides (AMPs) are evolutionarily conserved molecules that play a central role in innate immunity and represent a promising class of candidates against antimicrobial resistance. However, their clinical application is hindered by low stability in physiological conditions, susceptibility to proteolytic degradation, and dose-dependent toxicity. In this study, we evaluated the structural properties, antimicrobial activity, and therapeutic safety of two scorpion-derived AMP analogs, StigA25 and StigA31, in both free and encapsulated into poly (lactic-co-glycolic acid) (PLGA) nanoparticles. Both peptides displayed high stability in the range of temperature and pH investigated, retained activity in serum, and higher α-helical content in the presence of negatively charged lipid vesicles. Encapsulation efficiency above 95% in PLGA nanoparticles contributed to preserving the antimicrobial activity, enhanced bactericidal kinetics, and reduced hemolytic effects of tested peptides. In vitro assays indicated safety toward mammalian cells, while electron scanning microscopy revealed microbial membrane disruption. In a murine polymicrobial sepsis model, pretreatment with free and nanoformulated peptides effectively reduced bacterial load and attenuated inflammatory responses. Immunomodulation response included decreased leukocyte migration, myeloperoxidase activity, and pro-inflammatory cytokines TNF-α and IL-1β, with superior performance of the peptides in formulation. Collectively, these findings demonstrate enhanced therapeutic potential for scorpion-derived AMP analogs-loaded PLGA nanoparticles, providing a dual antimicrobial and anti-inflammatory strategy for severe infections such as sepsis, and support further development of AMP-based nanomedicines.