Pleurotus species are found to be among the most efficient lignocellulolytic types of white-rot fungi. Rice is the main grain cultivated in the extreme south of Brazil. Defatted rice bran and straw are by-products of low aggregate value. Soft rush (Juncus effusus) is a common native plant also very abundant in the region. In the present work, we evaluated changes in substrate composition after growth of two white-rot fungal species: Pleurotus ostreatus and Pleurotus sajor-caju, aiming to increase protein content and digestibility from substrates through solid fermentations and obtain edible mushrooms of high aggregate value. For that, defatted rice bran, defatted rice straw and soft rush were utilized as substrate. The influence of the variables thermal treatment temperature of substrate, substrate moisture and concentration were evaluated on the protein content, digestibility and biological efficiency. The highest protein enrichment of rice bran in P. sajor-caju-fermented medium was due the fact that there was no fructification in these media, while for the P. ostreatus-fermented medium, part of the synthesized protein was converted into mushrooms. The highest protein enrichments were verified in medium with 80% moisture and 25% soft rush (47.1% using P. ostreatus and 49.0% using P. sajor-caju). A higher digestible protein increase was obtained for both species in media with 70% moisture and 25% soft rush.