Viola is the largest genus in Violaceae plant family. Many Viola species belonging to the Violaceae family are considered as important herbs used in Asian traditional medicines. Viola is also known for its unique natural production of cyclotides. Here we report the first discovery of cyclotides from three Viola species in the northern mountainous region in Vietnam which have not been studied so far at this particular geographical latitude. A total of twelve cyclotides were discovered from these three Viola species including four new and eight known cyclotides. To gain information on their bioactivities and structure-activity relationships, these cyclotides were screened against human cancer and red blood cells on cytotoxicity assays, followed by structural characterization using nuclear magnetic resonance spectroscopy. This is the first study to unravel cyclotides found in Vietnam focusing on the Violaceae plant family. New sequences arise from this study have significantly expanded the cyclotide sequence diversity through comparison with other cyclotides discovered from the Asian region. Characterization of the bioactivity of these novel cyclotides has also provided valuable information for future drug design applications.