The attine ant symbiosis is characterized by ancient but varying degrees of diffuse co-evolution between the ants and their fungal cultivars. Domesticated fungi became dependent on vertical transmission by queens and the ant colonies came to rely on their symbiotic fungus for food and thus, indirectly, on fungal enzymes to break down the plant material brought in by the ants as fungal substrate. The more than 210 extant fungus-growing ant species differ considerably in colony size, social complexity and substrate-use. Only the derived leaf-cutting ants are specialized on using fresh leaves as garden substrate, whereas the more basal genera use leaf litter, insect feces and insect carcasses. We hypothesized that enzyme activity of fungal symbionts has co-evolved with substrate use and we measured enzyme activities of fungus gardens in the field to test this, focusing particularly on plant decomposing enzymes. We further obtained gene sequences coding for specific enzymes and used them to reconstruct the fungal symbiont phylogeny and to compare the trees obtained with those known from sequence information of genes that have no specific link to enzyme function. Differences in fungus garden enzyme activity across ant genera could indeed be partially explained by substrate differences. This implies that fungal enzyme activity has likely coevolved with the genus- or species-specific substrates that the ants use to manure their fungus garden. Plant decomposing enzymes are thus not only essential for the symbiosis in general, but have contributed specifically to the evolution of the symbiosis.