Macrofungi are among the most promising sources of biologically active natural products with nutritional qualities and therapeutic values. In this work, the nutritional value of nine species of wild macrofungi from Ibague-Tolima (Colombia) was evaluated. In addition the antioxidant, antimicrobial and cytotoxic activities of an ethanol:water (70:30) extract of wild basidiomata were evaluated. The wild mushrooms’ nutritional potential showed that the genusPleurotusandLentinushave the best protein percentages, with 18.4% and 18.5%. The nine extracts evaluated managed to stabilize the two radicals evaluated; however, lower IC50was found forPhellinus gilvusandGanoderma australeextracts. The results showed thatTrametes coccinea,Pleurotus floridanusandGanoderma australeextracts were the most effective as antimicrobials, with high inhibition percentages againstStaphylococcus aureus,Escherichia coli,Pseudomonas aeruginosaandKlebsiella pneumoniae. Antifungal activity results againstRhizopus oryzae,Penicilliumsp. andAspergillus nigershowed that the nine extracts were effective at the concentrations tested. Considering cell viability against isolated leukocytes, seven of the nine extracts showed percentages higher than 50% of cell viability. This research describes the nutritional value of nine wild macrofungi in Colombia and their potential for antimicrobial, cytotoxic and antioxidant activity.