The effects of local anesthetics (LAs), namely, lidocaine (LDC), prilocaine (PLC), mepivacaine (MPV), bupivacaine (BPV), procaine (PC), and tetracaine (TTC), on the steady-state transmembrane conductance induced by the cis-side addition of the antifungal polyene macrolide antibiotic, nystatin (NYS), in planar lipid bilayers were studied. The addition of TTC to model membranes comprising DOPC and cholesterol (33 mol%) led to a nearly twenty-fold increase in the steady-state NYS-induced membrane conductance. BPV slightly enhanced the channel-forming activity of polyene. LDC, PLC, MPV, and PC did not affect the NYS-induced transmembrane current. We concluded that the effects of LAs on the channel-forming activity of NYS were in agreement with their effects on the elastic properties of model membranes. The ability of aminoamide LAs to promote calcein leakage from large unilamellar DOPC-vesicles was decreased in the following order: BPV >> LDC ≈ PLC ≈ MPV. LDC, PLC, and MPV produced a graded leakage of fluorescent marker from liposomes, up to 10-13%. A initial sharp jump in fluorescence after the introduction of BPV was attributed to the solubilization of liposomes and the formation of mixed DOPC:BPV-micelles. Differential scanning microcalorimetry (DSC) of large unilamellar DPPC-vesicles showed that the main transition temperature (Tm) is continuously decreased upon increasing concentrations of TTC. A sharp drop in the enthalpy of the transition at higher TTC concentrations indicated a formation of anesthetic/lipid mixed micelles. In contrast to TTC, PC slightly decreased Tm, broadened the DSC signal and did not provoke vesicle-to-micelle transition. Both the calcein leakage and DSC data together with the results of measurements of threshold voltages that are required to cause the lipid bilayer breakdown might indicate an alteration in the curvature lipid packing stress, induced by BPV and TTC. The data presented here lend support to a lipid-mediated mode of LAs action on NYS pores via an alteration in curvature stress near the trans-mouth. Similar results were obtained for several lipid pores, formed by polyene amphotericin B, lipopeptide syringomycin E, and the peptides magainin and melittin. This finding further developed the concept of non-specific regulation of lipid pores by LAs. In conclusion, the combination of nystatin with LAs could be a novel treatment for efficient therapy of superficial and mucosal candidiasis.