Polysaccharide-based nanocarriers offer a novel delivery system for improving the stability, controlled release, and biological functionality of plant-derived bioactive materials. Olive leaf extract (OLE), rich in polyphenolic compounds with antioxidant and other bioactive properties, is limited by low stability and bioavailability. In this study, OLE-loaded alginate–chitosan nanoparticles were prepared using ionotropic gelation–polyelectrolyte complexation (IG-PEC) method, and their physicochemical properties, cytotoxic behavior, and potential prebiotic effects were evaluated. The resulting nanoparticles (232–237 nm) exhibited uniform spherical morphology, negative zeta potentials, and improved colloidal stability. Free OLE demonstrated concentration-dependent and selective cytotoxicity toward A549 and MCF-7 cancer cells, while exhibiting lower toxicity toward normal fibroblasts. In contrast, unloaded and OLE-loaded nanoparticles (1X, 2X) showed low cytotoxicity, suggesting superior biocompatibility of the polysaccharide nanocarrier. Notably, cultures supplemented with OLE-loaded nanoparticles showed a trend toward higher probiotic growth compared to free OLE, indicating a potential prebiotic effect and improved microbial tolerance to polyphenols during extended exposure. These findings highlight the advantages of polysaccharide-based nanoencapsulation for both stabilizing bioactive materials and supporting favorable microbial responses. The developed OLE nanocarriers may serve as a promising platform for nutraceutical, biomedical, and functional food applications.