The objective of the study was to design, synthetize and characterize poly(lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) with entrapped fish oil (FO) for possible application in a cutaneous wound healing. Morphology of NPs was evaluated by transmission electron microscopy. Antimicrobial characteristics were tested using the disk diffusion method and plate count method, and cytotoxicity was evaluated by the MTT assay. Fish oil (y) was released from PLGA NPs within the time interval (x) of 96 h according to equation y = 6.2 + 0.914x. PLGA-FO NPs did not affect growth of Staphylococcus aureus or methicillin-resistant S. aureus (MRSA) strains. No cytotoxic effect of the tested NPs on the keratinocyte cell line was observed for concentration of 1 μg/ml. PLGA-FO NPs represent an interesting alternative for wound healing due to an excellent biocompatibility and unique release profile of FO, despite their lack of antimicrobial efficiency.