AimsThe aim of this study was to improve production of pentaene 32,33-didehydroroflamycoin (DDHR) in Streptomyces durmitorensis MS405 strain to obtain quantities sufficient for in depth analysis of antimicrobial properties. Methods and ResultsThrough classical medium optimization conditions for stable growth, DDHR production within 7days of incubation was established. Yields of 215mgl(-1) were achieved in shake flask experiments in complex medium with mannitol as the primary carbon source. DDHR had poor antibacterial activity with minimal inhibitory concentrations (MIC) of 400gml(-1) for Staphylococcus aureus and Bacillus subtilis, while MIC of 70gml(-1) was determined for Candida albicans. Using flow cytometry and fluorescent microscopy, it was demonstrated that DDHR induced membrane damage in C.albicans followed by cell death. Combination studies with known antifungal nystatin showed that DDHR is a promising agent for the development of novel antimycotic treatments potentially less toxic for human cells. ConclusionsPentaene didehydroroflamycoin has no antibacterial activity but can be further developed for the application in antifungal therapy. Significance and Impact of the StudyThis study is the first report on the stable and production in high yields of a novel pentaene family that acts on Candida cell membranes and can be used in combination with known antifungals. Polyenes are still antifungal antibiotics of choice, and therefore, isolation and production of new lead structures are highly significant.