Filipin III is a polyene macrolide antibiotic derived from Streptomyces filipinensis. It exhibits antifungal activity against Candida utilis with an MIC of 0.3 mg/L and Saccharomyces cerevisiae with an MIC of 0.4 mg/L by interacting with membrane sterols, leading to alterations in membrane structure. Filipin III has a strong affinity for cholesterol and exhibits fluorescent properties, with the potential to be used as a valuable tool for detecting and quantifying cholesterol in biological membranes.