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 genus Pleurotus and Lentinus have the best protein percentages, with 18.4% and 18.5%. The nine extracts evaluated managed to stabilize the two radicals evaluated; however, lower IC50 was found for Phellinus gilvus and Ganoderma australe extracts. The results showed that Trametes coccinea, Pleurotus floridanus and Ganoderma australe extracts were the most effective as antimicrobials, with high inhibition percentages against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. Antifungal activity results against Rhizopus oryzae, Penicillium sp. and Aspergillus niger showed 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.