An extensive analysis of the anti-parasite activity of bis-acridine compounds was undertaken using a focused library of 18 structurally diverse compounds. The compound library was screened for in vitro bioactivity against Plasmodium falciparum, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Leishmania major, Schistosoma mansoni, Entamoeba histolytica and Trichomonas vaginalis, and for cytotoxicity in two mammalian cell lines. Structure-activity-relationships (SAR) were derived for each target, and demonstrated the influence of the bis-acridine linker and terminal heterocycle on potency and cytotoxicity. Certain bis-acridine compounds were active at nanomolar concentrations against Plasmodium falciparum and Trypanosoma brucei. Low therapeutic indices could be mitigated by structural modification of the linker, such that certain bis-acridine compounds demonstrated equivalent or better therapeutic profiles than existing clinical treatments. No one compound was effective against all parasite targets. New parasite targets were identified including, Schistosoma mansoni, Entamoeba histolytica and Trichomonas vaginalis. A novel class of bis-aza-acridine compounds was effective against a broad range of parasite targets. Our approach illustrates the usefulness of screening focused compound libraries against multiple, related parasite targets.Sandler Family Supporting Foundation, NIAID, Wellcome Trust, James Irvine Foundation