Bioactivity-guided fractionation of the methanolic extract of Psorothamnus polydenius yielded the new chalcone 2,2',4'-trihydroxy-6'-methoxy-3',5'-dimethylchalcone (2), together with six other known compounds, 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (1), dalrubone (3), demethoxymatteucinol (4), eriodictyol (5), and photodalrubone (6a and 6b). This is the first report of chalcones in P. polydenius. The extracts and isolated compounds were tested in vitro for their antiprotozoal activity against Leishmania donovani and Trypanosoma brucei. Chalcones 1 and 2 and dalrubone (3) exhibited leishmanicidal (IC(50) 5.0, 7.5, and 7.5 microg/mL, respectively) and trypanocidal (IC(50) 6.3, 6.8, and 21.6 microg/mL, respectively) properties. Dalrubone (3) displayed 6-fold selectivity for axenic L. donovani parasites over Vero cells. Furthermore, treatment of L. mexicana-preinfected macrophages with chalcones 1 and 2 and dalrubone (3) (12.5, 12.5, and 25 microg/mL, respectively) reduced the number of infected macrophages by at least 96% while posing no toxicity to the host cell.