Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by progressive inflammation of the intra- and extrahepatic bile ducts and a high association with inflammatory bowel disease. Since bacterial and viral stimuli are thought to contribute to the pathogenesis of autoimmune diseases, our study aims to investigate the effects of synthetic pathogenic agonists on the cytotoxic function of cluster of differentiation (CD)56⁺natural killer (NK) cells and CD8⁺ T cells in PSC. A total of 17 PSC patients, 18 autoimmune hepatitis (AIH) patients, and 14 healthy controls (HCs) were included in this study. Using multicolor flow cytometry, we analyzed cytotoxic activity (CD107a assay) and secretion of interferon-gamma and tumor necrosis factor-alpha in peripheral CD56 ⁺ NK-cell subsets and CD8⁺ T cells after in vitrostimulation with synthetic bacterial (Pam3CSK, lipopolysaccharide [LPS], CpG-ODN-2216, and flagellin) and viral agonists (polyinosinic: polycytidylic acid [poly(I: C)], poly(I: C)-high molecular weight/LyoVec^™, 5’ppp-dsRNA/LyoVec^™, and R837). Compared with AIH patients and HCs, CD56⁺ NK-cell subsets from PSC patients showed reduced cytotoxicity both at baseline and upon in vitrostimulation with synthetic bacterial/viral agonists. Notably, the percentage of unstimulated CD107a ⁺ CD56^dim CD16⁺NK cells positively correlated with clinical serum parameters in PSC. In addition, the percentage of CD107a⁺ CD8⁺ cells was significantly reduced after synthetic bacterial/viral stimulation in PSC patients. Finally, in PSC patients, production of pro-inflammatory cytokines was markedly reduced in CD56^highCD16⁻ NK cells upon in vitrostimulation with Pam3CSK and LPS, respectively. This hyporesponsiveness of CD56⁺NK cells and CD8⁺ T cells may contribute to the development of hepatic inflammation, representing a hallmark of PSC pathogenesis.