Nanofibers are a class of nanomaterial with specific physicochemical properties and characteristics making them quite sought after and investigated by researchers. Lipopeptide biosurfactant (LPB) formulation properties were previously established in wound healing. LPB were isolated from in vitro culture of Acinetobacter junii B6 and loaded on nanofibers formulation produced by electrospinning method with different ratios of carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), and Poloxamer. Numerous experimental control tests were carried out on formulations, including physicochemical properties which were evaluated by using dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), morphology study by scanning electron microscopy (SEM), and thermal stability. The best nanofibers formulation was obtained by the electrospinning method, with a voltage of 19.8 volts, a discharge capacity of 1cm/h, a cylindrical rotating velocity of 100rpm, and a needle interval of 7cm from the cylinder, which continued for 7hours. The formulation contained 2% (w/v) CMC, 10% (w/v) poloxamer, 9% (w/v) PVA, and 5% (w/v) LPB. This formula had desirable physicochemical properties including spreadability, stability, and uniformity with the particle size of about 590nm.