The bark of <i>Rhus verniciflua</i> Stokes (RVS) has been used to treat cancer in Korean herbal medicine. When we screened for PD-1 and CTLA-4 immune checkpoint inhibitors (PD-1/PD-L1 CTLA-4/CD80) from around 800 herbal extracts using competitive Enzyme-Linked Immunosorbent Assay (ELISA), we found that RVS blocked both the PD-1/PD-L1 and the CTLA-4/CD80 interactions. To identify the active compounds from RVS, we performed bioactivity-guided fractionation, and the ethyl acetate (EtOAc) fraction of RVS proved to be the most effective at blocking the PD-1/PD-L1 and CTLA-4/CD80 interactions. In addition, we isolated and identified 20 major compounds in the EtOAc fraction of RVS and then examined the blocking effects of these 20 compounds on PD-1/PD-L1 and CTLA-4/CD80. Among them, four compounds [eriodictyol (<b>7</b>) > fisetin (<b>9</b>) > quercetin (<b>18</b>) > liquiritigenin (<b>13</b>)] blocked the interaction of PD-1/PD-L1 on competitive ELISA. In addition, four different compounds [protocatechuic acid (<b>2</b>) > caffeic acid (<b>19</b>) > taxifolin (<b>5</b>) > butin (<b>6</b>)] blocked the interaction of CTLA-4/CD80. Our findings suggest that RVS and its components could be used as a potential immune checkpoint inhibitor blockade and could be developed for immuno-oncological therapeutics.