The aim of this study was to evaluate biodegradable poly(lactide-co-glycolide) nanoparticles as potential nano-delivery systems for the food antifungal compound natamycin. Natamycin-loaded nanoparticles were prepared at various ratios polymer/antifungal by the nanoprecipitation technique, resulting in nano-size particles (80–120 nm) with a narrow distribution and a spherical morphology. Complexation of natamycin with PLGA and active participation to the nanoparticle formation were evidenced by a mean diameter reduction of 10–30 nm, although encapsulation levels remained low due to the zwitterionic and partially hydrophilic nature of natamycin. Physical state analyses highlighted the presence of natamycin in an amorphous or molecularly dispersed state within the polymeric matrix. This translates into high availability of free antifungal molecules reflected in burst release and fast in vitro release kinetics rates as well as enhanced antifungal performance against the model food yeast Saccharomyces cerevisiae, offering a potential benefit for antifungal protection compared with the commercially available natamycin products.