Testicular tumors although rare tumors affect the young male population at the most productive age. Seminoma make up 50% of all diagnosed testicular tumors. Diagnosis, therapy and follow-up include invasive procedures that affect the quality of life of each patient. Classical tumor markers from the blood have their place in the diagnosis and monitoring of patients with seminoma, but they also have their limitations in sensitivity as well as specificity. Ejaculate, as a representative of liquid biopsies, is a non-invasive source of information for patients with seminoma and the isolation of cell free DNA (cfDNA) from the ejaculate of patients with seminoma is a potential source of markers for screening and monitoring patients with seminoma. In this study, we analyzed the methylation pattern of the OCT3/4 and NANOG genes in the ejaculate of patients with seminoma, preoperatively and postoperatively. We also analyzed the protein expression of the same genes in seminoma, GCNIS tissue and surrounding tissue with preserved spermatogenesis. A statistically significant difference in protein expression of OCT3/4 and NANOG and GCNIS was observed in relation to the surrounding tissue with preserved spermatogenesis. We did not prove a statistically significant difference in the methylation of the OCT3/4 and NANOG genes between preoperative and postoperative ejaculates of patients with seminoma, nor a statistically significant difference in the correlation relations between protein expression and methylation of cfDNA genes of the OCT3/4 and NANOG. To the best of our knowledge, this is the first study of cfDNA methylation OCT3/4 and NANOG gene in ejaculate of patients with testicular seminoma and may serve as an important source of information for further ejaculate research as an ongoing biopsy for screening and monitoring of patients with testicular tumors.