Developing drugs for brain infection by Naegleria fowleri is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to identify inhibitors that target an essential N. fowleri enzyme, sterol 14-demethylase (NfCYP51). A total of 124 compounds preselected in silico were tested against N. fowleri. Nine primary hits with EC50 ≤ 10 μM were phenotypically identified. Cocrystallization with NfCYP51 focused attention on one primary hit, miconazole-like compound 2a. The S-enantiomer of 2a produced a 1.74 Å cocrystal structure. A set of analogues was then synthesized and evaluated to confirm the superiority of the S-configuration over the R-configuration and the advantage of an ether linkage over an ester linkage. The two compounds, S-8b and S-9b, had an improved EC50 and KD compared to 2a. Importantly, both were readily taken up into the brain. The brain-to-plasma distribution coefficient of S-9b was 1.02 ± 0.12, suggesting further evaluation as a lead for primary amoebic meningoencephalitis.