The ethanolic extract of the stem bark of Croton heliotropiifolius Kunth (Euphorbiaceae) showed significant in vitro inhibition of acetylcholinesterase using a dilution spectrophotometric assay and antifungal activity against Candida albicans with a thin layer chromatography (TLC) bioautographic assay. In order to isolate the active compounds, bioassay-guided fractionation was undertaken using HPLC to localize the active compounds. Different zones of the HPLC-UV chromatogram were linked to acetylcholinesterase inhibition or to antifungal activities. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and HPLC-TOF-HRMS were used for the early identification of some of the compounds present. The targeted isolation of the active compounds was performed by medium pressure liquid chromatography (MPLC-UV) and further semi-preparative HPLC. Using this approach, nine compounds were isolated, one of them being a new indole alkaloid derivative. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, NMR, MS and HRMS.