Unique physiochemical and biological properties of nanofibers along with the choice of a wide variety of materials for both fabrication and tunable release patterns make nanofibers an ideal option for drug delivery. Loading antibacterial agents into nanofibers has attracted great deal of attention. Whilst there are several studies focusing on applying new generations of antibacterial materials, antibiotics are still the gold standard in clinical applications. Therefore, we aimed at introducing antibiotic-loaded nanofiber substrates with potential for topical skin delivery applications, reduced consumption of antibiotics and increased storage time. We applied Amoxicillin (AMX) as a model drug with low solubility and detected the presence of AMX in our nanofibers using FTIR and Raman spectroscopy. AMX-loaded Pullulan (Pull) nanofibers proved to maintain the antibacterial properties of the AMX drug after electrospinning, and to preserve the antibacterial properties for at least 8 months storage. The release trend can be tuned from burst release in mono-layer AMX:Pull nanofibers to sustained release if sandwiching the Pull layer between two hydrophobic electrospun layers (e.g. PLGA biopolymer). The AMX-loaded Pull construct can be considered as a novel nanofibrous solid dispersion of a poorly water-soluble drug for efficient topical application of antibiotics in wound healing and skin treatments.