ABSTRACT This study aimed to evaluate the antitumor activity of fractions rich in indole alkaloids extracted from the leaves of the species Tabernaemontana catharinensis . The chemical composition was analyzed using high‐resolution mass spectrometry (HRMS), which identified nine known indole alkaloids, including voacangine, voacangine hydroxyindolenine, ibogamine, and affinisine. In the subsequent step, both the extract and the fractions were tested against the human colorectal carcinoma cell line HCT116. Cytotoxicity assays showed that the total alkaloid fraction (AF) exhibited selectivity against this tumor cell line (IC 50 28.49 µg mL −1 ), while preserving the viability of the non‐tumor MRC5 cell line (IC 50 60.35 µg mL −1 ) after 24 h of treatment. Additionally, it exhibited moderate cytotoxicity in the brine shrimp ( Artemia salina ) assay (LC 50 367.33 ± 2.45 µg mL −1 ). The results obtained in this study suggest that the alkaloids present in the fractions and subfractions synergistically have potential for the development of selective chemotherapeutic agents for colorectal carcinoma.