The present paper deals with the preparation and annotation of a surfactin(s) derived from a culture of the endophytic bacterium Bacillus 15F. The LC-MS analysis of the acetonitrile fraction confirmed the presence of surfactins Leu/Ile7 C15, Leu/Ile7 C14 and Leu/Ile7 C13 with [M+H]+ at m/z 1036.6895, 1022.6741 and 1008.6581, respectively. Various concentrations of the surfactin(s) (hereafter referred to as surfactin-15F) were used to reduce the adhesion of Staphylococcus epidermidis S61, which served as a model for studying antibiofilm activity on polystyrene surfaces. Incubation of Staphylococcus epidermidis S61 with 62.5 µg/ml of surfactin-15F resulted in almost complete inhibition of biofilm formation (90.3 ± 3.33%), and a significant reduction of cell viability (resazurin-based fluorescence was more than 200 times lower). The antiadhesive effect of surfactin-15F was confirmed by scanning electron microscopy. Surfactin-15F demonstrated an eradication effect against preformed biofilm, causing severe disruption of Staphylococcus epidermidis S61 biofilm structure and reducing viability. The results suggest that surfactins produced by endophytic bacteria could be an alternative to synthetic products. Surfactin-15F, used in wound dressings, demonstrated an efficient treatment of the preformed Staphylococcus epidermidis S61 biofilm, and thus having a great potential in medical applications.