Three polyene antibiotics, mediomycins A (1), B (2), and clethramycin (3), were isolated from Streptomyces mediocidicus ATCC23936. Their structures were elucidated through extensive NMR study coupled with chemical reactions and MS/MS fragmentation analysis. All three compounds are linear polyenes consisting of a conjugated oxo-triene group and a hexaene moiety. Compounds 1 and 2 are new polyenes. All three compounds demonstrated a broad spectrum of antifungal activity in vitro.