This research reports about the development by electrospinning of fiber-based films made of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) derived from fermented fruit waste, so-called bio-papers, with enhanced antimicrobial performance. To this end, different combinations of oregano essential oil (OEO) and zinc oxide nanoparticles (ZnONPs) were added to PHBV solutions and electrospun into mats that were, thereafter, converted into homogeneous and continuous films of 130 µm. The morphology, optical, thermal, mechanical properties, crystallinity, and migration into food simulants of the resultant PHBV-based bio-papers were evaluated and their antimicrobial properties were assessed against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in both open and closed systems. It was observed that the antimicrobial activity decreased after 15 days due to the release of the volatile compounds, whereas the bio-papers filled with ZnONPs showed high antimicrobial activity for up to 48 days. The electrospun PHBV biopapers containing 2.5 wt% OEO + 2.25 wt% ZnONPs successfully provided the most optimal activity for short and long periods against both bacteria. Nanomaterials 2020, 10, 506 2 of 26 studied, including the poly(3-hydroxybutyrate) (PHB or P3HB) homopolyester and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The latter shows lower crystallinity and melting point as well as greater flexibility, which broadens its industrial applicability [4,5]. In order to confer active properties to PHAs, several substances can be incorporated into the biopolymers such as essential oils (EOs) and inorganic or metal nanoparticles (MNPs) [6,7].EOs are the product of the secondary metabolism of plants, separated from the aqueous phase, which is formed by various volatile components such as terpenes, alcohols, acids, esters, epoxies, aldehydes, ketones, amines, and sulphides, among others [8]. This wide range of compounds are responsible for the strong biological activity and also the antioxidant, antimicrobial, antifungal, and antiviral properties of plants [9]. In addition, EOs are biologically safe and have a low risk of causing resistance in pathogens [10]. In addition, they are classified as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA) [11]. Among them, oregano essential oil (OEO) is one of the most important and it is widely used in the pharmaceutical, food, and cosmetics industries [12]. It comes from the genus Origanum that belongs to the Lamiaceae family [13], which is constituted by more than 38 monoterpenoids [14]. Their main active compounds are carvacrol, thymol, p-cymene, and γ-terpinene, which are responsible for its high antimicrobial and antioxidant activities [15,16]. These active compounds have been particularly related to the inhibition of G− bacteria such as Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Salmonella typhimurium (S. typhimurium) and G+ bacteria including Staphylococcus aureus (S. aureus), Listeria mo...