| Summary: | The leaves of <i>Pyrola rotundifolia</i> L. were extracted in the mixed solvent of methanol/acetone/water (2:2:1, <i>v</i>/<i>v</i>/<i>v</i>) and investigated for their phytochemical analysis and biological activity. Total phenolic and flavonoid contents were determined spectrophotometrically. A high content of phenols (208.35 mg GAE/g of dry extract), flavonoids (38.90 mg QE/g of dry extract) and gallotannins (722.91 GAE/g of dry extract) was obtained. Ultra-high performance liquid chromatography diode array detector tandem mass spectrometry (UHPLC–DAD–MS) allowed for the detection of 23 major peaks at 254 nm. The extract was analyzed for its antioxidant capacity using 2,2-diphenyl-1-picryl-hydrazyl (DPPH<sup>•</sup>) and 2,2′-azinobis[3-ethylbenzthiazoline]-6-sulfonic acid (ABTS<sup>•+</sup>) radical scavenging, metal chelating power and β-carotene-linoleic acid bleaching assays. The examined extract showed moderate radical scavenging and chelating activity, and good inhibiting ability of linoleic acid oxidation (EC<sub>50</sub> = 0.05 mg/mL) in comparison to standards. The cytotoxic effect in increasing concentration on five types of leukemic cell lines was also investigated using trypan blue vital staining. It was found that the analyzed extract induced the apoptosis of all the tested cell lines. Our findings suggest that the leaves of <i>P. rotundifolia</i> are a source of valuable compounds providing protection against oxidative damage, hence their use in traditional medicine is justified.
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