Natural and environmentally benign electrospun polymeric nanofibers (NFs) with distinctive features, unique physiochemical and biological properties, and high susceptibility to carry non-toxic antibacterial cargoes, e.g., ferrocenyl-based compounds, have emerged as a neo-tool for killing bacteria. Here, the antibacterial FcLR-Chit/Pull NFs were fabricated through three steps: 1) synthesis of FcLR-Chit via the attachment of chitosan to Ferrocenyl Lawesson’s Reagent (FcLR), 2) blending of the FcLR-Chit at various concentrations with the natural polymer of pullulan, and 3) electrospinning. The successfully characterized FcLR-Chit/Pull NFs with a homogenous distribution of FcLR-Chit inside the pullulan matrix were thermally stable up to 270–290 ºC and mechanically stable with an increased elongation at break (EAB) (30.0–42.2%) and decreased tensile strength (TS) (1.1–2.3 MPa) compared to the pure pullulan NFs with the EAB of 19.0% and TS of 3.6 MPa. Pure pullulan NFs and FcLR-Chit/Pull NFs (0.1 wt%, 1.0 wt%, and 5.0 wt%) showed insignificant cytotoxicity on the fibroblast cell lines (L929). The antibacterial activity was detected in the trend of FcLR-Chit/Pull NFs (5.0 wt%)>FcLR-Chit powder (5.0 wt% in water)>FcLR-Chit/Pull NFs (1.0 wt%)>FcLR powder (5.0 wt% in water)>FcLR-Chit/Pull NFs (0.1 wt%)>chitosan powder (5.0 wt% in water)> pure pullulan NFs. The FcLR-Chit/Pull NFs (5.0 wt%) with an average diameter of 365 nm, insignificant cytotoxicity on L929 cells within 24 h and 48 h, and boosted antibacterial activity with the inhibition zone diameter of 1.95 mm against Gram-negative E. Coli and 1.76 mm against Gram-positive S. aureus bacteria were chosen as the optimal nanofibrous candidate for the future bactericidal applications.