The search for new substances with cytotoxic activity against various cancer cells, especially cells that are very resistant to currently used chemotherapeutic agents, such as melanoma cells, is a very important scientific aspect. We investigated the cytotoxic effect of Chelidonium majus, Mahonia aquifolium and Sanguinaria canadensis extracts obtained from different parts of these plants collected at various vegetation stages on FaDu, SCC-25, MCF-7, and MDA-MB-231 cancer cells. Almost all the tested extracts showed higher cytotoxicity against these cancer cells than the anticancer drug etoposide. The highest cytotoxicity against the FaDu, SCC-25, MCF-7 and MDA-MB-231 cancer cell lines was obtained for the Sanguinaria candensis extract collected before flowering. The cytotoxicity of extracts obtained from different parts of Chelidonium majus collected at various vegetation stages was also evaluated on melanoma cells (A375, G361 and SK-MEL-3). The highest cytotoxic activity against melanoma A375 cells was observed for the Chelidonium majus root extract, with an IC50 of 12.65 μg/mL. The same extract was the most cytotoxic against SK-MEL-3 cells (IC50 = 1.93 μg/mL), while the highest cytotoxic activity against G361 cells was observed after exposure to the extract obtained from the herb of the plant. The cytotoxic activity of Chelidonium majus extracts against melanoma cells was compared with the cytotoxicity of the following anticancer drugs: etoposide, cisplatin and hydroxyurea. In most cases, the IC50 values obtained for the anticancer drugs were higher than those obtained for the Chelidonium majus extracts. The most cytotoxic extract obtained from the root of Chelidonium majus was selected for in vivo cytotoxic activity investigations using a Danio rerio larvae xenograft model. The model was applied for the first time in the in vivo investigations of the extract’s anticancer potential. The application of Danio rerio larvae xenografts in cancer research is advantageous because of the transparency and ease of compound administration, the small size and the short duration and low cost of the experiments. The results obtained in the xenograft model confirmed the great effect of the investigated extract on the number of cancer cells in a living organism. Our investigations show that the investigated plant extracts exhibit very high cytotoxic activity and can be recommended for further experiments in order to additionally confirm their potential use in the treatment of various human cancers.