Hepatitis B virus (HBV) is one of the most widespread viral infections in the world and poses a significant global public health problem. The implementation of effective vaccination programs has resulted in a significant decrease in the incidence of acute hepatitis B. Nevertheless, there is still a need for as many effective anti-HBV drugs as possible. In this study, the role of pu-erh tea extracts (PTE) against HBV was analyzed in vitro by using a stably HBV-transfected cell line HepG2 2.2.15. The MTT assay showed that PTE and its active components (tea polyphenols, theaflavins, and theanine) presented low cytotoxicity. ELISA analysis revealed that PTE effectively reduced the secretion of HBeAg, but any one of the active components alone showed weaker efficacy, suggesting that the anti-HBV activity of PTE might be a synergetic effect of different components. RT-PCR and luciferase assay showed that PTE suppressed HBV mRNA expression while leaving four HBV promoter transcriptional activities unchanged. Fluorescence quantitative PCR results demonstrated that PTE dramatically diminished HBV DNA produced in cell supernatants as well as encapsidated DNA in intracellular core particles. Finally, PTE significantly reduced intracellular reactive oxygen species (ROS) level. This study is the first to demonstrate that PTE possesses anti-HBV ability and could be used as a potential treatment against HBV infection with an additional merit of low cytotoxicity.