This work intended to prospect new phytase-producing organisms. In silico genomic analyses allowed the selection of twelve potential phytase-producing fungi. Based on gene sequence, it was possible to identify four well-defined groups of phytate-degrading enzymes: esterase-like, beta-propeller phytases (beta PP), phosphoglycerate mutase-like, and phytases of the histidine acid phosphatases (HAP) family. Analysis of the predicted genes encoding phytases belonging to the HAP family and beta PP phytases and in silico characterization of these enzymes indicated divergence among the catalytic activities. Predicted fungal beta PP phytases exhibited higher molecular mass (around 77 kDa) probably due to the epidermal growth factor-like domain. Twelve sequences of phytases contained signal peptides, of which seven were classified as HAP and five as beta PP phytases, while ten sequences were predicted as phytases secreted by non-classical pathways. These fungi were grown in liquid or semi-solid medium, and the fungal enzymatic extracts were evaluated for their ability to hydrolyze sodium phytate at 50 degrees C and pH ranging from 2.0 to 9.0. Seven fungi were identified as phytase producers based on phosphate release under enzyme assay conditions. Results obtained from in silico analyses combining experimental enzymatic activities suggest that some selected fungi could secrete beta PP phytases and HAP phytases.