Fast-growing Zygomycetes, most notably Rhizopus oligosporus, are traditionally used in many food fermentations, for example, for soybean tempeh production. R. oligosporus is considered to belong to the Rhizopus microsporus group. Certain R. microsporus strains have been reported to produce either the pharmaceutically active rhizoxins or the highly toxic rhizonins A and B. In this study was investigated the formation of secondary metabolites by R. microsporus, R. oligosporus, and Rhizopus chinensis grown on a wide range of different semisynthetic and natural substrates. Liquid chromatography, combined with photodiode array detection and high-resolution mass spectrometric techniques, was used to identify secondary metabolites. Growth on maize, brown rice, and Pharma agar gave both the highest amounts and the maximum diversity of rhizoxins and rhizonins. Rhizoxins were produced by all four R. microsporus strains, whereas only one strain produced rhizonins. The six R. oligosporus and four R. chinensis strains investigated did not produce any of these two classes of metabolites.