The development of wound-dressing materials has attracted significant research interests in recent years. With the advancement of nanofabrication, the application of nanoparticles (NPs) in drug delivery systems has become feasible. However, most existing work focuses on incorporation of metal, metal/semimetal oxide, or organic particles into nanofiber scaffolds. There has been a lack of work on the incorporation of drug-encapsulated polymeric particles into nanofiber scaffolds. In this study, gentamicin-encapsulated poly(lactic-co-glycolic acid) (PLGA) NPs were synthesized via a double emulsion solvent evaporation method. Electrospinning was used to incorporate gentamicinencapsulated PLGA NPs into nanofiber scaffolds. Atomic force microscopy (AFM), dynamic light scattering, scanning electron microscopy (SEM), ultraviolet−visible spectroscopy (UV−vis), and an agar diffusion method were utilized to characterize the morphologies, release profiles, and antibacterial activities of various gentamicin-loaded PLGA NP-incorporated nanofiber scaffolds. The results indicated that the PLGA NPs had a spherical morphology with an average diameter of 130 nm. Purification of PLGA NPs was essential to eliminate the residual poly(vinyl alcohol) (PVA) and to prevent particle agglomeration. The purified PLGA NPs were uniformly and individually incorporated into the polyurethane (PU)/ poly(ethenyl oxide) (PEO) or PEO-only nanofiber scaffolds but nearly none into the PU-only fiber scaffolds. PEO served as a continuous phase in the PU/PEO mixture, which significantly improved the compatibility of PLGA NPs and PU, resulting in a well-dispersed distribution of PLGA NPs in the monolithic nanofiber scaffolds. Excellent antibacterial properties against Escherichia coli were found in both PU/PEO and PEO nanofiber scaffolds. This study of incorporating gentamicin-encapsulated PLGA NPs into fiber scaffolds provides insights for achieving successful incorporation of drug-encapsulated polymeric NPs into fiber scaffolds. This offers a promising microfabrication technology for delivery of therapeutic molecules with controlled release for biomedical applications.