Testicular germ cell tumours (TGCTs) are the most common malignancies in Caucasian young men and their incidence has increased over the past decades. However, a non-invasive test allowing an early diagnosis of TGCT often proves inaccurate. We have previously shown that two Cancer-Testis Antigens (CTA), namely MAGE-A4 and NY-ESO-1, were expressed by TGCT. As exfoliation of carcinoma in situ (CIS) cells or tumour germ cells from testis into seminal fluid can occur, here we studied the expression of the 2 CTA in semen smears of patients with testicular cancer in comparison with healthy men. Using semen smears from healthy controls (n = 65) and patients diagnosed for testicular tumour (n = 57) and immunological staining, we observed expression of MAGE-A4 and NY-ESO-1 proteins in seminal fluid exfoliated cells. We found a highly statistically significant difference in the ratios of stained cells to the total number of round cells between testicular cancer patients and healthy controls. Multivariable analysis, including sperm parameters and immunostaining on sperm smears, shows the improvement. This technique can provide towards testicular cancer diagnosis when it is included in the current testing regime. However, the fact that expression of these markers was not restricted to foetal germ cells led to detection in the semen of a number of healthy subjects. Although the detection of these CTA could be useful to characterize the sub-type of individual TGCTs better, we stress here that the false positive rate precludes the exclusive employment of these CTA for the early detection of testicular neoplasia.