Influenza A virus is the major cause of epidemics and pandemics worldwide. In this study, virtual screening was used to identify compounds interacting with influenza A polymerase PB2 cap-binding domain (CBD). With a database of 21,351 small molecules, 28 candidate compounds were obtained and four of them (225, 225D1, 426 and 426Br) were found to bind directly to PB2 CBD by surface plasmon resonance (SPR). The evaluation of compounds 426Br and 225 indicated that they could bind to PB2 CBD and inhibit influenza virus at low micromolar concentration. They were predicted to bind the cap binding site of the protein by molecular modeling and were confirmed by SPR assay using PB2 CBD mutants. These two compounds have novel scaffolds and could be further developed into lead compound for influenza virus inhibition.