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.