Biological invasion is the successful spread and establishment of a species in a novel environment that adversely affects the biodiversity, ecology, and economy. Both invasive and non-invasive species of the Caulerpa genus secrete more than thirty different secondary metabolites. Caulerpin is one of the most common secondary metabolites in genus Caulerpa. In this study, caulerpin found in invasive Caulerpa cylindracea and non-invasive Caulerpa lentillifera extracts were analyzed, quantified, and compared using high-performance thin layer chromatography (HPTLC) for the first time. The anticancer activities of caulerpin against HCT-116 and HT-29 colorectal cancer (CRC) cell lines were also tested. Caulerpin levels were found higher in the invasive form (108.83 ± 5.07 µg mL−1 and 96.49 ± 4.54 µg mL−1). Furthermore, caulerpin isolated from invasive Caulerpa decreased cell viability in a concentration-dependent manner (IC50 values were found between 119 and 179 µM), inhibited invasion-migration, and induced apoptosis in CRC cells. In comparison, no cytotoxic effects on the normal cell lines (HDF and NIH-3T3) were observed. In conclusion, HPTLC is a quick and novel method to investigate the caulerpin levels found in Caulerpa extracts, and this paper proposes an alternative utilization method for invasive C. cylindracea due to significant caulerpin content compared to non-invasive C. lentillifera.