Fifty-eight Alternaria isolates representing 10 species or species-groups and 66 Penicillium isolates representing 18 species were examined for their growth response to the combined effects of water activity, temperature and pH in an extended Central Composite Design. Growth responses were recorded as colony diameter after one and two weeks of growth and analysed using different multivariate statistical analyses. The isolates, when analysed by Principal Component Analysis, clustered according to their genus and to some degree to species or species groups and not according to substratum as excepted. Soft Independent Modelling of Class Analogy and Response Surface Analysis showed that growth responses or growth profiles may be used as classification tool. Partial Least Squares Regression showed that a combination of two different media based on Dichloran Rose bengal Yeast Extract Sucrose agar incubated at two different temperatures were enough to get genus segregation and to some extent species segregation. The results also showed that water activity, temperature and pH interact strongly in their effect on growth rates and that the squared products (optima) of water activity, temperature and pH for each isolate were important for modelling the data sufficiently.