AbstractBalamuthia mandrillaris, a pathogenic free-living amoeba (FLA), causes cutaneous skin lesions as well as the brain-eating disease:Balamuthiagranulomatous amoebic encephalitis (GAE). These diseases, and diseases caused by other pathogenic FLA,Naegleria fowleriorAcanthamoebaspecies, are minimally studied from a drug discovery perspective; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Chemotherapies for CNS disease caused byB. mandrillarisrequire vast improvement. Current therapeutics are limited to a small number of drugs that were previously discovered in the last century throughin vitrotesting or identified after use in the small pool of surviving reports.Using our recently published methodology to identify potentially useful therapeutics, we screened a collection of 85 compounds that have previously been reported to have antiparasitic activity. We identified 59 compounds that impacted growth at concentrations below 220 μM. Since there is no fully annotated genome or proteome, we used RNA-Seq to reconstruct the transcriptome ofB. mandrillarisand locate the coding sequences of the specific genes potentially targeted by the compounds identified to inhibit trophozoite growth. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing.