Hepatitis B virus (HBV) infection is a major risk factor for chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC) worldwide. While multiple hepatitis B drugs have been developed, development of drug resistance during treatment or weak efficacies observed in some cases have limited their application. Therefore, there is an urgent need to develop better pharmacological agents for HBV-infected individuals. Here, we identified cetylpyridinium chloride (CPC) as a novel inhibitor of HBV. Using computational analysis of CPC-nucleocapsid proteins, microscale thermophoresis analysis of CPC binding to viral nucleocapsids, and in vitro nucleocapsid formation assays, we found that CPC interacts with dimeric viral nucleocapsid protein (known as core protein or HBcAg). Compared with other HBV inhibitors, such as benzenesulfonamide and sulfanilamide, CPC achieved significantly better reduction of HBV particle number in HepG2.2.15 cell line, a derivative of human HCC cells that stably expresses HBV. Taken together, our results show that CPC inhibits capsid assembly and leads to reduced HBV biogenesis. Thus, CPC is an effective pharmacological agent that can eliminate HBV particles.