Oral squamous cell carcinoma (OSCC) is a common head and neck cancer with low survival rates, especially in advanced stages, despite improved therapies. New developments show that immune checkpoint inhibitors (ICIs) are promising treatment options. A better understanding of immune suppression in OSCC could enable new therapeutic approaches and effective ICI combinations. The aim of this cross-sectional study was to investigate the significance of the differential expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), CD28 and their ligands CD80 and CD86 for the diagnosis and treatment of OSCC. To this end, mRNA expression was analysed by RT-PCR and compared in 65 healthy oral mucosa samples (NOM) and 104 OSCC samples. The expression of CTLA-4 (a soluble and membrane-bound isoform) was increased in OSCC by 1.72-fold ( = 0.004) and 6.88-fold ( < 0.001), respectively. There was no significant difference for CD28 ( = 0.283), nor for the soluble isoform of CD86 ( = 0.845). The membrane isoform of CD86 was increased in OSCC by a factor of 1.39 ( = 0.009) and CD80 by 6.11-fold ( < 0.001). The results show a significant association between CTLA-4, CD80 and membrane-bound CD86 expression and diagnosis. They could improve diagnostics in multi-marker approaches and serve as therapeutic targets for ICI strategies. In particular, the data indicate a stronger immunosuppressive role of CD80 compared to CD86 in a tumor tissue context, suggesting the exploration of anti-CTLA-4 and anti-CD80 antibody combinations in animal models.