This investigation concerns the design of poly(3-hydroxybutyrate) (PHB)-based nanofibrous hybrid materials containing zinc oxide nanoparticles (nano-ZnO) by means of two electro-hydrodynamic techniques, i.e., electrospinning of polymer/nano-ZnO solutions and the combination of electrospinning of polymer solutions with electrospraying of nano-ZnO dispersions. The analysis of the physical properties associated with precursory solutions was performed in order to understand the final morphology of the corresponding nanofibers. The obtained PHB/nano-ZnO mats showed uniform fiber morphology with an average porosity ca. 85 % with enhanced thermal stability compared to that of pristine PHB. Differential scanning calorimetry was also used to determine the influence of ZnO nanoparticles in the phase transitions of as-spun PHB nanofibers. Furthermore, the antibacterial performance against E. coli and S. aureus proved to be dependent on the elaboration technique, thus permitting the design of novel bacteriostatic or bactericidal PHB/nano-ZnO nanofibrous composites.