Melanoma is a serious form of skin cancer that begins in cells known as melanocytes. While it is less common than the other forms of skin cancer, melanoma is more dangerous because of its ability to spread to other organs more rapidly if it is not treated at an early stage. The number of people diagnosed with melanoma has increased over the last few decades. The most widely used treatments include surgery, chemotherapy, and radiation therapy. The search for new drugs to treat various cancers is one of the most important challenges of modern scientific research. Some isoquinoline alkaloids found in different plant species have strong cytotoxic effects on various cancer cells. We tested the effect of isoquinoline alkaloids and extracts obtained from various parts of Mahonia aquifolium collected in various vegetation seasons on human melanoma cancer cells and our data indicated that investigated extract induced significant reduction in cell viability of Human malignant melanoma cells (A375), human Caucasian malignant melanoma cell line (G361), and human malignant melanoma cell line (SKMEL3 cancer cell lines in a dose- and time-dependent manner. Differences in cytotoxic activity were observed for extracts obtained from various parts of Mahonia aquifolium. Significant differences were also obtained in the alkaloids content and cytotoxic activity of the extracts depending on the season of collection of plant material. Our investigations exhibit that these plant extracts can be recommended for further in vivo experiments in order to confirm the possibility of their use in the treatment of human melanomas.