Golden Ganoderma is a valuable species in the medicinal formula Luc Bao Linh Chi which has been used in traditional medicine in Vietnam. The golden Ganoderma mushroom sample discovered at Delonix regia tree in Hue city, Thua Thien Hue province was identified as Ganoderma colossus (Fr.) C.F. Baker. This study investigated the cytotoxicity of A549 lung cancer cells and the antibacterial activity of the aqueous extract from the fruiting body of golden Ganoderma. The extract at a concentration of 1 mg/mL showed the strongest inhibitory activity against strains of Bacillus subtilis (27.1 ± 0.36 mm), Staphylococcus aureus (14.2 ± 0.54 mm), Gram negative bacteria strains (Escherichia coli and Salmonella enteritidis) were weaker inhibited. The cytotoxic effect on lung cancer cells was determined by the MTT method [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide)]. The results showed that the extract at a concentration of 128 µg/mL had an inhibitory effect on A549 lung cancer cells of 86.36% with an IC50 value of 9.21 ± 0.8 µg/mL compared to the positive control Elipcitine of 0.34 ± 1.13 µg/mL.