Humans are widely exposed to a great variety of mycotoxins and their mixtures. Therefore, it is important to design strategies that allow prioritizing mycotoxins based on their toxic potential in a time and cost-effective manner. A strategy combining in silico tools (Phase 1), including an expert knowledge-based (DEREK Nexus(R)(,) Lhasa Limited, Leeds, UK) and a statistical-based platform (VEGA QSAR (c), Mario Negri Institute, Milan, Italy), followed by the in vitro SOS/umu test (Phase 2), was applied to a set of 12 mycotoxins clustered according to their structure into three groups. Phase 1 allowed us to clearly classify group 1 (aflatoxin and sterigmatocystin) as mutagenic and group 3 (ochratoxin A, zearalenone and fumonisin B1) as non-mutagenic. For group 2 (trichothecenes), contradictory conclusions were obtained between the two in silico tools, being out of the applicability domain of many models. Phase 2 confirmed the results obtained in the previous phase for groups 1 and 3. It also provided extra information regarding the role of metabolic activation in aflatoxin B1 and sterigmatocystin mutagenicity. Regarding group 2, equivocal results were obtained in few experiments; however, the group was finally classified as non-mutagenic. The strategy used correlated with the published Ames tests, which detect point mutations. Few alerts for chromosome aberrations could be detected. The SOS/umu test appeared as a good screening test for mutagenicity that can be used in the absence and presence of metabolic activation and independently of Phase 1, although the in silico-in vitro combination gave more information for decision making.