Two new alkaloids, phranisines A–B (1–2), along with two known compounds, N-p-Coumaroyl serotonin (3) and N-p-coumaroyl-tryptamine (4), were isolated from the roots of Phragmites australis. The structures of 1–4 were established on the basis of extensive spectroscopic. The absolute configuration of compounds 1–2 were identified through quantum-chemical electronic circular dichroism (ECD) calculation compared with their experimental CD. All the isolated compounds were tested for their cytotoxic activities against HeLa and MCF-7 human cancer cell lines, and compounds 2–4 showed moderate cytotoxic activities against HeLa cell lines with IC50 values ranging from 13.2 to 18.6 μM. © 2021 Informa UK Limited, trading as Taylor & Francis Group.