A variety of secondary metabolites are formed and compounds released during the bioconversion of lignocellulosic biomass by white-rot fungi that can affect the nutritional value and acceptance of the biomass by ruminants. Changes in pH, ergosterol content, fibre and metabolites composition of wheat straw (WS) incubated with either Ceriporiopsis subvermispora or Lentinula edodes for up to 8 weeks were investigated. With increases in mycelium content, significant decreases in absolute amount of hemicellulose, acid detergent lignin and, to a lesser extent, cellulose were observed in both fungal treatments. Acidification mainly occurred within the first four weeks of incubation, coinciding with the largest changes in metabolites profile. Diverse compounds, including organic acids and soluble sugars increased or decreased with C. subvermispora and L. edodes treatment. None of the thirty-four common mycotoxins analyzed were detected in WS after 8 weeks of fungal incubation. These results provide important information for application of fungal treated WS that might affect animal acceptance and performance.