The exploration of fungal diversity in Cesar, Colombia represents a strategic priority due to the need to identify bioactive compounds that support the development of sustainable biotrade in pharmaceutical, nutraceutical, and agrochemical sectors, as well as to provide resources for ecosystem conservation and restoration in the context of climate change. In this framework, the present study aimed to preliminarily assess the biotechnological potential of Auricularia nigricans and Muscodor sp. collected in Pueblo Bello, Cesar, Colombia. Taxonomic characterization of both species was conducted through macroscopic, microscopic, and molecular analyses. For A. nigricans, reducing sugars, total carbohydrates, and total phenolic content were quantified to establish an initial biochemical profile. In Muscodor sp., liquid and solid-state fermentations using mycelium were performed to evaluate extracellular protein concentration and enzymatic activity. Additionally, the mycelium’s bioremediation capacity was assessed in aqueous methylene blue solutions. Results showed that A. nigricans exhibited a high total carbohydrate content and moderate levels of total phenolics, suggesting potential nutraceutical applications. Crude enzymatic extracts from Muscodor sp. presented low protein concentration and limited enzymatic activity; however, the mycelium achieved up to 76% degradation of methylene blue, demonstrating bioremediation potential against dye contaminants. This study constitutes the first report of taxonomic and biochemical characterization of A. nigricans and Muscodor sp. in the Cesar, Colombia, providing valuable insights for regional bioprospecting and laying the groundwork for future research focused on the identification of metabolites with biotechnological potential and the integration of fungal resources into bioeconomy and ecological restoration strategies.