The transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases. A primary therapeutic target for TSE intervention has been a protease-resistant form of prion protein known as PrPSc or PrP-res. In vitro testing of mouse scrapie-infected cell cultures has identified many PrP-res inhibitors that also have activity in vivo. Here we identify 32 new inhibitors of two strains of mouse scrapie PrP-res. Furthermore, to investigate the species-specificity of these and other PrP-res inhibitors, we have developed a high-throughput cell culture assay based on Rov9 cells chronically-infected with sheep scrapie. Of 32 inhibitors of murine PrP-res that were also tested in the Rov9 cells, only six showed inhibitory activity against sheep PrP-res. The three most potent inhibitors of both murine and ovine PrP-res formation (with 50% inhibition at ≤5_M) were tannic acid, pentosan polysulfate and Fe(III) deuteroporphyrin 2,4-bisethyleneglycol. The latter two have anti-mouse scrapie activity in vivo. These results identify new inhibitors of murine and ovine PrP-res formation and reinforce the idea that compounds effective against PrP-res from one species or strain cannot be assumed to be active against others.