Nucleos(t)ide analog (NA) therapy has proven effective in treating chronic hepatitis B. However, NAs frequently result in viral relapse after the cessation of therapy. This is because NAs cannot fully eliminate the viral episomal covalently closed circular DNA (cccDNA) in the nucleus. In this study, we identified small molecular compounds that control host factors related to viral replication using in silico screening with simulated annealing based on bioinformatics for protein-ligand flexible docking. Twelve chemical compound candidates for alpha-glucosidase (AG) inhibitors were identified from a library of chemical compounds and used to treat fresh human hepatocytes infected with HBV. They were then monitored for their anti-viral effects. HBV replication was inhibited by one candidate (1-[3-(4-tert-butylcyclohexyl)oxy-2-hydroxypropyl]-2,2,6,6-tetramethylpiperidin-4-ol) in a dose-dependent manner. This compound significantly reduced ccc DNA production, compared to Entecavir (p < 0.05), and had a lower anti-AG effect. Gene expression analysis and structural analysis of this compound showed that its inhibitive effect on HBV was via interaction with Sp1. The nuclear transcription factor Sp1 acts on multiple regions of HBV to suppress HBV replication. Identifying candidates that control nuclear transcription factors facilitate the development of novel therapies. Drugs with a mechanism different from NA are promising for the elimination of HBV.