Naegleria fowleri is the causative agent of primary amebic meningoencephalitis (PAM) in humans. Individuals are infected when amoeba-contaminated water forcefully enters the nose. N. fowleri migrates from the nasal cavity to the frontal lobe of the brain, where it feeds on brain tissue. The urgency for novel therapeutics is underscored by the 98% fatality rate of PAM. In this study, we developed a secondary assay to evaluate the ability of compounds to inhibit N. fowleri replication. We found that of the compounds tested, the inhibitory concentrations required for metabolic inhibition were similar to the concentrations required for inhibition of cellular replication. We additionally developed a secondary assay to evaluate the ability of compounds to inhibit N. fowleri-mediated destruction of a mammalian cell monolayer. Protease inhibitors were not found to be effective in preventing N. fowleri-mediated monolayer destruction, however, cytoskeleton inhibitors were effective. The cytopathogenicity assay was optimized for high-throughput screening and used to screen a novel 80-compound library. The library was counter-screened against N. fowleri trophozoites alone and in a cytotoxicity assay. Ultimately, we identified four lead compounds for further development. The fluorescent nature of the compounds allowed them to be localized within N. fowleri trophozoites. Visualization revealed the compounds localize to a non-nuclear compartment in the cytoplasm. In the final portion of this project, we utilized high-throughput screening to identify promising drug targets for N. fowleri. We screened three novel libraries against N. fowleri trophozoites. We identified 26 nanomolar inhibitors against N. fowleri, 16 of which had not previously been reported for activity against N. fowleri. Analysis of all hit compounds <5 µM from all high-throughput screening campaigns conducted against N. fowleri allowed us to generate a list of potential drug targets for further validation and structure-based drug design. Histone deacetylase and aurora kinases were the most populous hit compound targets. Further investigation of active nucleoside analogs led us to propose a pathway for the previously undescribed purine salvage pathway in N. fowleri. The purine salvage pathway provides a promising avenue for further N. fowleri drug target development.