Although antimicrobial peptides have been shown to inactivate viruses through disruption of their viral envelopes, clinical use of such peptides has been hampered by a number of factors, especially their enzymatically unstable structures. To overcome the shortcomings of antimicrobial peptides, peptoids (sequence-specific N-substituted glycine oligomers) mimicking antimicrobial peptides have been developed. We aimed to demonstrate the antiviral effects of antimicrobial peptoids against hepatitis B virus (HBV) in cell culture. The anti-HBV activity of antimicrobial peptoids was screened and evaluated in an infection system involving the HBV reporter virus and HepG2.2.15-derived HBV. By screening with the HBV reporter virus infection system, three (TM1, TM4, and TM19) of 12 peptoids were identified as reducing the infectivity of HBV, though they did not alter the production levels of HBs antigen in cell culture. These peptoids were not cytotoxic at the evaluated concentrations. Among these peptoids, TM19 was confirmed to reduce HBV infection most potently in a HepG2.2.15-derived HBV infection system that closely demonstrates authentic HBV infection. In cell culture, the most effective administration of TM19 was virus treatment at the infection step, but the reduction in HBV infectivity by pre-treatment or post-treatment of cells with TM19 was minimal. The disrupting effect of TM19 targeting infectious viral particles was clarified in iodixanol density gradient analysis. In conclusion, the peptoid TM19 was identified as a potent inhibitor of HBV. This peptoid prevents HBV infection by disrupting viral particles and is a candidate for a new class of anti-HBV reagents.