The general objective of this research was to gain insight into the mechanisms of action of the antimicrobial cyclic lipopeptide fengycin on model membranes. Total internal reflection fluorescence (TIRF) microscopy was used to visualize and analyse lipid phase separation within supported lipid bilayers (SLBs) of various compositions. The initial research investigated the influence of protocol parameters on fluorescent probe distribution and lipid domain appearance within SLBs. The influence of sonication and extrusion during liposomal preparations, choice of solid support containment, and ratio of unsaturated and saturated phospholipids (DOPC:DPPC) in SLBs were assessed. Liposomal solutions passed through an extruder after sonication saw a decrease in TR-DHPE aggregates. Bilayers composed of a 3:1 DOPC:DPPC with 0.8% mol TR-DHPE produced bilayers with even fluorescence, distinct phase separations, and limited photobleaching. Using this optimized protocol, ergosterol was incorporated into the bilayers and domain redistribution was evaluated after the addition of fengycin. SLBs containing higher concentrations of ergosterol were more tolerant to changes induced by fengycin. Following the analysis of these ternary SLBs, extracted lipids from molds with different fengycin sensitivity were use in the preparation of SLBs. Bilayers composed of Alternaria solani lipids were most tolerant to fengycin. A. solani also contained the highest level of ergosterol compared to Fusarium sambucinum and Pythium sulcatum. The redistribution of lipids phases it these membranes could also be attributed to the readsorption of phospholipid-fengycin micelles produced during bilayer solubilization. Overall, the results of these works confirm that ergosterol plays a key role in the antimicrobial activity of fengycin. An increase in ergosterol content strongly correlates with an increase in ordered phases, potentially promoting membrane insolubility as seen in surfactant resistant membranes.