Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme's substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase (or hydroxy acid oxidase 1, HAO1) is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors.