In the present study, tailor-made ethylene vinyl alcohol copolymer (EVOH) fibers containing different amounts of antimicrobial silver ions and nanoparticles were developed by electrospinning and subsequent thermal annealing. The morphology of the fibers was examined by scanning and transmission electron microscopy and thermal properties were characterized by differential scanning calorimetry. Speciation and controlled release of silver from the fibers was monitored by anodic stripping voltammetry and energy dispersive X-ray spectroscopy. Before aging, 100 % of the silver recovered from the electrospun structures was in ionic form to be instantly released in contact with moisture with varying temperature-dependent kinetics. Thermal annealing of the fibers at 100 °C for 1, 2, and 4 days prompted the gradual transformation of 70, 93–94, and 98–99 % of the total silver into nanoparticles homogeneously distributed along the fibers, which were mostly retained within them, producing a substantial decrease in their release capacity. Speciation and release profiles from the fibers were correlated with their antibacterial performance against Listeria monocytogenes and Salmonella enteric. This study is a step forward in the understanding of silver-based electrospun antimicrobial polymers and puts forth the suitability of EVOH for the development of targeted delivery systems in a number of applications.