<jats:p>The present work aimed to analyze the alkaloid content of the ethanolic extract of <jats:italic>Tabernaemontana catharinensis</jats:italic> (Apocynaceae family) and its fractions as well as to evaluate their antioxidant and anticholinesterasic activities. The analyses of the ethanolic extract of <jats:italic>T. catharinensis</jats:italic> by mass spectrometry allowed identifying the presence of the alkaloids 16‐epi‐affinine, coronaridine‐hydroxyindolenine, voachalotine, voacristine‐hydroxyindolenine, and 12‐methoxy‐n‐methyl‐voachalotine, as well as an alkaloid with <jats:italic>m/z</jats:italic> 385.21 whose spectrum suggests a derivative of voacristine or voacangine. The extract and its alkaloid rich fractions showed antioxidant activity, especially those that contain the alkaloid <jats:italic>m/z</jats:italic> 385.21 or 16‐epi‐affinine with DPPH scavenging activity (IC<jats:sub>50</jats:sub>) between 37.18 and 74.69 <jats:italic>μ</jats:italic>g/mL. Moreover, the extract and its fractions exhibited anticholinesterasic activity, particularly the fractions characterized by the presence of 12‐methoxy‐n‐methyl‐voachalotine, with IC<jats:sub>50</jats:sub> = 2.1 to 2.5 <jats:italic>μ</jats:italic>g/mL. Fractions with 16‐epi‐affinine combined good antioxidant (IC<jats:sub>50</jats:sub> = 65.59 to 74.69 <jats:italic>μ</jats:italic>g/mL) and anticholinesterasic (IC<jats:sub>50</jats:sub> = 7.7 to 8.3 <jats:italic>μ</jats:italic>g/mL) activities, representing an option for further studies aimed at treating neurodegenerative diseases.