Biodegradable nanocomposites were prepared by adding ZnO nanoparticles to bacterial polyester poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) via solution casting technique. The morphology, thermal, mechanical, antibacterial, barrier, and migration properties of the nanocomposites were analyzed. The nanoparticles were uniformly dispersed within PHBV without the aid of coupling agents, and acted effectively as nucleating agents, raising the crystallization temperature and the level of crystallinity of the matrix while decreasing its crystallite size. A gradual rise in thermal stability was found with increasing ZnO loading, since the nanofillers hinder the diffusion of volatiles generated during the decomposition process. The nanocomposites displayed superior stiffness, strength, toughness, and glass transition temperature, whereas they displayed reduced water uptake and oxygen and water vapor permeability compared to the neat biopolymer, related to the strong matrix-nanofiller interfacial adhesion attained via hydrogen bonding interactions. At an optimal concentration of 4.0 wt % ZnO, the tensile strength and Young's and storage moduli showed a maximum that coincided with the highest crystallinity and the best barrier properties. PHBV/ZnO films showed antibacterial activity against human pathogen bacteria, and the effect on Escherichia coli was stronger than on Staphylococcus aureus. The overall migration levels of the nanocomposites in both nonpolar and polar simulants dropped upon increasing nanoparticle content, and were well below the limits required by the current normative for food packaging materials. These sustainable nanomaterials with antimicrobial function are very promising to be used as containers for beverage and food products as well as for disposable applications like cutlery or overwrap films.