Prions are infectious protein particles known to cause prion diseases. The biochemical entity of the pathogen is the misfolded prion protein (PrP^Sc) that forms insoluble amyloids to impair brain function. PrP^Sc interacts with the non-pathogenic, cellular prion protein (PrP^C) and facilitates conversion into a nascent misfolded isoform. Several small molecules have been reported to inhibit the aggregation of PrP^Sc but no pharmacological intervention was well established thus far. We, here, report that acylthiosemicarbazides inhibit the prion aggregation. Compounds <b>7x</b> and <b>7y</b> showed almost perfect inhibition (EC₅₀ = 5 µM) in prion aggregation formation assay. The activity was further confirmed by atomic force microscopy, semi-denaturing detergent agarose gel electrophoresis and real-time quaking induced conversion assay (EC₅₀ = 0.9 and 2.8 µM, respectively). These compounds also disaggregated pre-existing aggregates <i>in vitro</i> and one of them decreased the level of PrP^Sc in cultured cells with permanent prion infection, suggesting their potential as a treatment platform. In conclusion, hydroxy-2-naphthoylthiosemicarbazides can be an excellent scaffold for the discovery of anti-prion therapeutics.