Stephania rotunda (Menispermaceae), a creeper commonly found in the mountainous areas of Cambodia, has been mainly used for the treatment of fever and malaria. Thus, the aim of this study is to investigate the chemical composition and antiplasmodial activity of different samples of S. rotunda and compare their antiplasmodial activity with their alkaloid content. Sixteen samples from different parts (roots, stem, and tuber) of S. rotunda were collected from four regions of Cambodia (Battambang, Pailin, Siem Reap, and Kampot). Reversed-phase HPLC was used to determine the content of three bioactive alkaloids (cepharanthine, tetrahydropalmatine, and xylopinine). These three alkaloids have been found in all samples from Battambang and Pailin (samples I–IX), whereas only tetrahydropalmatine was present in samples from Siem Reap and Kampot (samples X–XVI). The analyzed extracts were evaluated for their antiplasmodial activity on W2 strain of Plasmodium falciparum. Among them, 13 extracts were significantly active with inhibitory concentration 50 (IC50) from 1.2 to 3.7 µg/mL and 2 extracts were moderately active (IC50 = 6.1 and 10 µg/mL, respectively), whereas sample XI was not active (IC50 = 19.6 µg/mL). A comparison between antiplasmodial activity and concentration of the three bioactive alkaloids in S. rotunda extracts has been realized. Copyright © 2012 John Wiley & Sons, Ltd.