This work aimed to isolate microorganisms from the digestive system of the fish Prochilodus lineatus and identify them biochemically. Among the five isolated colonies denominated as BI1, BI2, BI3, BE1 and LE1, four bacteria and a yeast were identified microscopically. The bacteria BI1 and BE1 fermented the medium of mannitol agar showing yellow staining, indicating positive for Staphylococcus coagulase, on the other hand the BI3, a Gram-negative bacteria, presented pink staining, indicating negative for Staphylococcus coagulase. LE1 yeast was able to grow on Eosin/Lactose agar medium, proving its ability to ferment lactose. Recent studies have evaluated the importance of Gram-negative bacteria in the production of cellulolytic enzymes for the hydrolysis of biomass. Similarly, lactose fermenting yeasts were studied for the production of bioethanol.