The polyene antifungal antibiotic nystatin confers its biological activity by forming pores in the membranes of target cells. Exposure of only one side of the membrane to nystatin is more relevant than two-side exposure because in vivo antibiotic molecules initially interact with cell membrane from the exterior side.The effect of flavonoids and styryl dyes on the steady-state conductance induced by a cis-side addition of nystatin was investigated by using electrophysiological measurements on artificial membranes. The assessment of changes in membrane dipole potential by dipole modifiers was carried out by their influence on K+-nonactin (K+-valinomycin) current. The alterations of the phase segregation scenario induced by nystatin and flavonoids were observed via confocal fluorescence microscopy.The introduction of phloretin, phlorizin, biochanin A, myricetin, quercetin, taxifolin, genistin, genistein, and RH 421 leads to a significant increase in the nystatin-induced steady-state transmembrane current through membranes composed of a mixture of DOPC, cholesterol and sphingomyelin (57:33:10mol%). Conversely, daidzein, catechin, trihydroxyacetophenone, and RH 237 do not affect the transmembrane current. Three possible mechanisms that explain the observed results are discussed: changes in the membrane dipole potential, alterations of the phase separation within the lipid bilayer, and influences of the dipole modifiers on the formation of the lipid mouth of the polyene pore.Most likely, changes in the monolayer curvature in the vicinity of trans-mouth of a nystatin single-length channel prevail over alterations of dipole potential of membrane and the phase segregation scenarios induced by dipole modifiers.