Primary amoebic meningoencephalitis (PAM) is >97% fatal disease caused by the brain-eating amoeba, Naegleria fowleri. The amoeba can infect immunocompetent individuals when it enters the nasal cavity and begins feeding on the nasal epithelium and olfactory nerves as it travels towards the frontal lobe of the brain. In this study, we developed a high-throughput assay to identify active compounds that inhibit amoebic growth. With this assay, we screened a library of amidines and diamidines that were known to be active against other infectious diseases such as Trypanosoma spp. and Leishmania species. We found that the bis-benzimidazole amidines and diamidines were the most potent compounds against the pathogenic N. fowleri. Additionally, we screened a library of FDA-approved compounds to identify the potential of repurposing drugs for PAM. In this study, we identified posaconazole to be among the fastest compounds that inhibit amoebic growth in vitro. In addition, In the murine PAM model we found 33% of Naegleria-infected mice treated with 3 doses of posaconazole survived. Moreover, when testing posaconazole in combination with azithromycin, we found a 40% increased survival of mice when compared to the fluconazole-azithromycin combination. From the results of this study, we propose to include posaconazole in PAM treatments instead of fluconazole. In the last portion of this project, we developed resistance to a potent bis-benzimidazole amidine, DB 2518, to elucidate a potential mechanism of action. We characterized multiple phenotypic differences to compare with the genotypic variations identified through whole-genome sequencing. We hypothesize from the decreased size of trophozoites and number of lysosomes in the resistant clones, that ion binding, lyase or hydrolase mutated genes may be likely targets for the bis-benzimidazole, DB 2518.