The influence of alternative carbon sources as inducers of -galactosidase and -fructofuranosidase by filamentous fungi (that are, Aspergillus aculeatus, Chrysonilia sitophila, Gliocladium virens, Aspergillus fumigatus and Trichoderma longibrachiatum), recently isolated from Brazil's Atlantic Forest biome has been investigated. The greatest levels of intracellular β-galactosidase activity were obtained using orange peel waste (56.31 U/mL) with A. aculeatus, rice straw (22.57 U/mL) with G. virens, sorghum straw (16.48 U/mL) with C. sitophila, and passion fruit peel with either A. fumigatus (17.26 U/mL) or T. longibrachiatum (17.53 U/mL). The most effective intracellular -fructofuranosidase activity was obtained by A. aculeatus using trub (409.46 U/mL) or passion fruit peel (44.59 U/mL). Thus, alternative carbon sources, such as orange peel and trub, exhibit great potential as inducers for the production of these enzymes. Such fungal isolates from the Atlantic Forest of Paraná, Brazil are promising candidates for generating significant amounts of β-galactosidase and -fructofuranosidase using abundant and inexpensive agro-industrial substrates.