Juglone (5-hydroxy-1,4-naphthoquinone) (J) is a naphthoquinone structured allelochemical that is mostly found in the roots, leaves, nut-hulls, bark, and wood of walnut (Juglans regia). In this study, the biocompatibility, mechanical, thermal, chemical, morphological, and antimicrobial properties of the poly(lactic acid) (PLA) (w/v)/J (10, 20, 30 mg) electrospun scaffolds were investigated. Based on the results of the study, it was shown that juglone addition increased the antimicrobial properties of the scaffolds against the Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), compared to the neat PLA film after 24 h of contact time. According to the tensile test results, the addition of J made the scaffolds more flexible but decreased the mechanical strength. The cytotoxicity properties of the J-added scaffolds demonstrated a toxic behavior on the first day of incubation. However, with an increase in the J ratio, the fibroblast cell metabolic activity increased for all incubation periods.