Two prenyltransferases from Aspergillus ruber control the echinulin biosynthesis via exceptional sequential prenylations. EchPT1 catalyzes the first prenylation step, leading to preechinulin. The unique EchPT2 attaches, in a consecutive prenylation cascade, up to three dimethylallyl moieties to preechinulin and its dehydro forms neoechinulins A and B, resulting in the formation of at least 23 2- to 4-fold prenylated derivatives. Confirming these products in fungal extracts unravels the unprecedented catalytic relevance of EchPT2 for structural diversity.