Prion diseases are associated with the conformational conversion of the physiological form of cellular prion protein (PrP(C)) to the pathogenic form, PrP(Sc). Compounds that inhibit this process by blocking conversion to the PrP(Sc) could provide useful anti-prion therapies. However, no suitable drugs have been identified to date. To identify novel anti-prion compounds, we developed a combined structure- and ligand-based virtual screening system in silico. Virtual screening of a 700,000-compound database, followed by cluster analysis, identified 37 compounds with strong interactions with essential hotspot PrP residues identified in a previous study of PrP(C) interaction with a known anti-prion compound (GN8). These compounds were tested in vitro using a multimer detection system, cell-based assays, and surface plasmon resonance. Some compounds effectively reduced PrP(Sc) levels and one of these compounds also showed a high binding affinity for PrP(C). These results provide a promising starting point for the development of anti-prion compounds.