Two new cytotoxic trisoxazole macrolides, thiomycalolides A (2) and B (3), have been isolated from a marine sponge Mycale sp. The structures were determined to be glutathione adducts of mycalolides A and B by interpretation of spectral data and chemical transformation. Thiomycalolides A and B are highly cytotoxic against P388 murine leukemia cells.