Lichens are a promising source of good anticancer agents. However the study on their bioactivities remains limited, especially in Vietnam. In this study, three extracts of lichen Usnea undulata Stirt. including n-hexane, acetone and methanol were investigated for their cytotoxic activity on two human cancer cell lines (MCF-7, NCI-H460) and normal fibroblasts by sulforhodamine B (SRB) assay. The results showed that the n-hexane extract exhibited the highest inhibition activity with IC50 5.260.13 µg/mL for MCF-7 and 6.830.64 µg/mL for NCI-H460, followed by acetone extract with IC50 66.441.37 µg/mL and 92.141.49 µg/mL, respectively. The methanol extract possessed weak cytotoxic effect against MCF-7 and NCI-H460 (IC50 values more than 100 µg/mL). The n-hexane extract demonstrated interesting activity particularly on MCF-7 as good selectivity index was recorded (SI > 3). The phytochemical study on n-hexane and acetone extracts led to the isolation of four compounds including usnic acid (1), methyl orsellinate (2), methyl β-orsellinate (3), 7-hydroxyl-5-methoxy-6-methylphtalide (4) in which compound (4) was isolated the first time in such species. Their structures were elucidated by NMR analysis and compared with literature data.