A series of 15 alkanediamide-linked bisbenzamidines and related analogs were synthesized and tested in vitro against two Trypanosoma brucei (Tb) strains: T. b. brucei (Tbb) and T. b. rhodesiense (Tbr), two Plasmodium falciparum (Pf) strains: a chloroquine-sensitive strain (NF54) and a chloroquine-resistant strain (K1), Trypanosoma cruzi (Tc), and Leishmania donovani (Ld). The in vitro cytotoxicity was determined against rat myoblast cells (L6). Seven compounds (5, 6, 10, 11, 12, 14, 15) showed high potency toward both strains of Tb and Pf with the inhibitory concentrations for 50% (IC₅₀) in the nanomolar range (IC₅₀ = 1-96 nM). None of the tested derivatives was significantly active against Tc or Ld. Three of the more potent compounds (5, 6, 11) were evaluated in vivo in mice infected with the drug-sensitive (Lab 110 EATRO and KETRI 2002) or drug-resistant (KETRI 2538 and KETRI 1992) clinical isolates of T. brucei. Compounds 5 and 6 were highly effective in curing 100% mice infected with the drug-sensitive strains, including a drug-resistant strain KETRI 2538, but were ineffective against KETRI 1992. Thermal melting of DNA and molecular modeling studies indicate AT-rich DNA sequences in the minor grove as possible binding sites for these compounds. Several of the tested compounds are excellent leads for the development of improved antiparasitic agents.