Antimicrobial materials have become relevant for local therapies preventing microbial resistance induced by systemic antibiotic treatments. This work reports the development of electrospun poly(lactic acid) (PLLA) nanofiber membranes loaded with bovine lactoferrin (bLF) up to 20 wt%. The membranes present smooth and nondefective fibers with mean diameters between 717 ± 197 and 495 ± 127 nm, and an overall porosity of ≈80%. The hydrophobicity of the PLLA membranes is reduced by the presence of bLF. The release profile of bLF correlates with an anomalous transport model, with 17.7 ± 3.6% being released over 7 weeks. The nanofiber mats show no cytotoxicity on human skin fibroblasts and even promote cell proliferation after short exposure periods. Furthermore, the developed membranes display antifungal activity against Aspergillus nidulans by inhibiting spore germination and mycelial growth. These results evidence the strong potential of bLF-PLLA nanofiber membranes to be used as antifungal dressings.