Targeted cancer therapy is considered important in cancer treatment, and microenvironment including immunotherapy has recently emerged as an effective treatment for various types of cancer. Activation of tumor infiltrating lymphocyte-mediated immune responses through blockade of immune checkpoint markers such as programmed cell death-1 (PD-1) or its partner ligand PD-L1 has been fundamental to the development of clinically effective anti-cancer therapies. However, these PD-1 and PD-L1-targeting blocker-derived antibodies are expensive to produce, have poor tumor penetration, and can cause autoimmune side effects, which limits their application. Here we showed that small-molecule antagonists of the PD-1/PD-L1 interaction exhibit potent antitumor activity in vitro and in vivo by alleviating the PD-1/PD-L1-induced T cell activity exhaustion. PDI-1 and pentamidine bound with high affinity to purified human and mouse PD-1 or PD-L1 proteins, which was confirmed in an in vitro competitive ELISA assay, computational docking analysis and pull-down assay. Culturing activated human T cells with PDI-1 and pentamidine enhanced cytotoxicity against colon cancer as well as other cancers including lung, breast and melanoma cells and increased production of granzyme B, perforin, and inflammatory cytokines. Additionally, PDI-1 and pentamidine directly increased TCR-mediated NFAT activation in a PD-1/PD-L1-dependent manner. In a syngeneic mouse tumor model, PDI-1 and pentamidine reduced the growth of tumors derived from human PD-L1 transfected mouse cancer cells by increasing tumor-infiltrating CD8+ T cells and decreasing PD-L1 expression in tumor tissues. These results suggest that small molecule inhibitors of PD-1/PD-L1 may be effective as alternative or complementary immune checkpoint inhibitors in cancer immunotherapy.