The antimicrobial polypeptide e-poly( l -lysine) (e-PL) was electrostatically incorporated to poly(acrylic acid) (PAA)/poly(vinyl alcohol) (PVA) electrospun nanofibers. e-PL loading and distribution was assessed by infrared spectra, ζ-potential measurements and the primary amino reactive dye fluorescamine. Functionalized fibers with 485 ± 140 nm diameter, could be loaded with 0.57–0.74 g e-PL (g dressing)−1 that released at a constant rate of 5.4 ± 2.8 mg e-PL (g dressing day)−1. Such a dressings resulted in two orders of magnitude lower bacterial colonization than non-functionalized PAA-PVA after 14 days of incubation. Bacterial impairment was attributed to the damage of cell membranes and the formation of intracellular reactive oxygen species. e-PL functionalized nanofibers did not display cytotoxicity to human corneal epithelial cells, HCEpC, in 24 h MTT assays. However, the viability of rapidly growing tumoral HeLa cells decreased >50% under the same conditions. The prepared biocompatible nanofibrous dressings with durable antibacterial activity show potential application as wound dressings and other biomedical uses.