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Chaga mushroom (<i>Inonotus obliquus</i>) is a pathogenic fungus that grows mostly on birch species (<i>Betula pendula</i> Roth and <i>B. pubescens</i> Ehrh.) and has traditionally been used as an anticancer medicine. This study aimed to compare the chemical composition and cytotoxic activity of chagas growing on both <i>Betula</i> spp. on various cancer cell lines. The freeze-dried extracts contained triterpenes inotodiol, lanosterol betulin, and betulinic acid typical to conks growing on <i>Betula</i> species. The cytotoxic activity of chaga growing on <i>Betula pendula</i> and <i>B. pubescens</i> 80% ethanolic extracts against 31 human cancer cell lines was evaluated by a sulforhodamine B assay. Chaga extract showed moderate activity against all cancer cell lines examined; it did not result in high cytotoxicity (IC₅₀ ≤ 20 µg/mL). The strongest inhibitions were observed with chaga (growing on <i>B. pendula</i>) extract on the HepG2 and CAL-62 cell line and with chaga (from <i>B. pubescens</i>) extract on the HepG2 cell line, with IC₅₀ values of 37.71, 43.30, and 49.99 μg/mL, respectively. The chaga extracts from <i>B. pendula</i> exert somewhat stronger effects on most cancer cell lines studied than <i>B. pubescens</i> extracts, which can be attributed to a higher content of inotodiol in <i>B. pendula</i> extracts. This study highlights the potential of chaga as a source of bioactive compounds with selective anticancer properties. To the best of our knowledge, this study is the first investigation of the chemical composition of <i>I. obliquus</i> parasitizing on <i>B. pubescens</i>.