Immune exhaustion is a hallmark of ovarian cancer. Using multiparametric flow cytometry, the study aimed to analyze protein expression of novel immunological targets on CD3 T cells isolated from the peripheral blood (  = 20), malignant ascites (  = 16), and tumor tissue (  = 6) of patients with ovarian cancer (OVCA). The study revealed an increased proportion of effector memory CD8 T cells in OVCA tissue and malignant ascites. An OVCA-characteristic PD-1 CD8 T cell population was detected, which differed from PD-1 CD8 T cells by increased co-expression of TIGIT, CD39, and HLA-DR. In addition, these OVCA-characteristic CD8 T cells showed reduced expression of the transcription factor TCF-1, which may also indicate reduced effector function and memory formation. On the contrary, the transcription factor TOX, which significantly regulates terminal T cell-exhaustion, was found more frequently in these cells. Further protein and gene analysis showed that CD39 and CD73 were also expressed on OVCA tumor cells isolated from solid tumors (  = 14) and malignant ascites (  = 9). In the latter compartment, CD39 and CD73 were also associated with the expression of the "don't eat me" molecule CD24 on tumor cells. Additionally, ascites-derived CD24 EpCAM tumor cells showed a higher frequency of CD39 or CD73 cells. Furthermore, CD39 expression was associated with unfavorable clinical parameters. Expression of CD39 on T cells was upregulated through CD3/CD28 stimulation and its blockade by a newly developed nanobody construct resulted in increased proliferation (eFluor), activation (CD25 and CD134), and production of cytotoxic cytokines (IFN-γ, TNF-α, and granzyme-B) of CD8 T cells.