Acacia longifolia is one of the main plant species invading Portuguese dune ecosystems. Areas invaded by this exotic tree have reduced plant diversity and altered soil microbial processes and nutrient pools, but the impacts on microbial functional diversity in the soil have been little explored. Soil samples were collected in areas invaded by A. longifolia for more than 20 years, in areas invaded after 1995 and in non-invaded areas. Respiration responses to 20 different substrates were analysed, in order to assess the catabolic response profile (CRP) as a measure of microbial functional diversity. Five substrate groups were tested: amino acids, carbohydrates, carboxylic acids, plant litters, and plant polymers. CRP clearly discriminated between the three different areas. Respiratory responses to the individual substrates a-ketoglutaric acid, oxalic acid, starch, citric acid, and xylose and to the groups of amino acids and plant polymers were similar in both invaded areas and different in the non-invaded. The responses to tartaric acid, gallic acid, fumaric acid, Cistus litter, and Acacia litter were the same in long- and non-invaded areas, but different from recently invaded areas. The duration of invasion, carbon (C) content, nitrogen (N) content, C/N ratio, pH, and litter quantity explained 39.6% of the variance of catabolic responses. It is concluded that invasion by A. longifolia has substantial effects on the catabolic diversity of the soil microbial communities. These effects may have wider implications for nutrient cycling and ecosystem-level processes and for the invasibility of the system.