The mycelial submerged culture of the Colombian mushroom Humphreya coffeata, the production of exopolysaccharides (EPS), and the evaluation of the supernatant components in terms of bioactivity were investigated. Three carbon sources, three concentrations of carbon and nitrogen sources, and three pH values were evaluated. The optimal source was lactose (over sucrose and glucose), and the maximal biomass and EPS concentrations (15.5 g/L and 6.9 g/L, respectively) were obtained using lactose at 50 g/L, yeast extract 10 g/L, and an initial pH of 4.5. The cytotoxic, proliferative, genotoxic, and antioxidant activities were evaluated in lyophilized culture filtrates. Cytotoxicity was analyzed on human nontumorigenic keratinocytes (HaCaT), two human epithelial cell lines of cervical cancer (HeLa and InBl), and one lymphoma cell line (Jurkat). None of the filtrate concentrations that showed a cytotoxic effect on Jurkat cells (250-2500 μg/mL) were toxic for control HaCat cells and cervical cancer (HeLa and InBl). Furthermore, DNA damage was observed at a high extract concentration (2500 μg/mL), and genoprotection was observed using the lowest concentration tested (250 μg/mL). In addition, antioxidant activity was observed via the superoxide anion and the DPPH scavenging. This suggests that H. coffeata extracts can repress initiation and/or break the chain of oxidation reactions.