Trimethoprim-sulfamethoxazole has been recommended as part of the standard package of care for people with HIV and AIDS in Africa. A similar antifolate combination, sulfadoxine-pyrimethamine, is now the first-line antimalarial drug in several of the African countries with the highest rates of HIV infection. We present evidence of Plasmodium falciparum cross-resistance between trimethoprim and pyrimethamine at the molecular level. The impact of trimethoprim-sulfamethoxazole on the efficacy of sulfadoxine-pyrimethamine needs to be assessed urgently, and alternative antimalarial treatment should be considered for people on trimethoprim-sulfamethoxazole prophylaxis.