A series of eleven biflavonoids containing amentoflavone and hinokiflavone derivatives from the Indian medicinal herb Selaginella bryopteris has been investigated for their antiprotozoal activity using in vitro assays against the K1 strain of Plasmodium falciparum, Leishmania donovani, Trypanosoma brucei rhodesiense and Trypanosoma cruzi. The highest antiprotozoal activity was displayed by 7,4′,7″-tri-O-methylamentoflavone which exhibited an IC 50 of 0.26μM. This compound showed no significant cytotoxicity (IC 50 >150μM) evaluated using L-6 cells. The strongest activity against Leishmania was detected for 2,3-dihydrohinokiflavone (IC 50 =1.6μM), whereas for Trypanosoma no significant activity was observed (IC 50 >12.5μg/mL for the extract). To evaluate the in vivo activity against Plasmodium of the most active compound, trimethylated amentoflavones were obtained by partial synthesis starting from amentoflavone. The synthesized mixture of trimethylated amentoflavones did not show activity in the Plasmodium berghei mouse model against female NMRI mice at 50mg/kg.