The oncogenic potential of viruses such as Epstein-Barr virus (EBV), Human Herpesvirus 8 (HHV-8), BK polyomavirus (BKPyV), and JC Polyomavirus (JCPyV) in breast cancer remains under debate. This study aimed to investigate the presence of these viruses in breast tissues using molecular techniques. Formalin-Fixed Paraffin-Embedded (FFPE) breast tissue samples from 108 patients with invasive breast carcinoma and 100 controls (fibrocystic or normal tissue) were analyzed by quantitative PCR. EBV DNA positive samples were further evaluated using EBER in situ hybridization (EBER-ISH). EBV DNA was detected in 4.6% of cancer tissues (5/108) and 1.0% of controls (1/100), with not statistically significant (p = 0.214). All EBV DNA positive malignant cases were over the age of 40 and diagnosed with Invasive Ductal Carcinoma (IDC). No EBER expression was observed in any sample. HHV-8, BKPyV, and JCPyV were not detected in any tissue. Our findings suggest a low prevalence of EBV DNA in breast cancer and no evidence of other DNA viruses. While no causal relationship was established, the presence of EBV exclusively in older patients with invasive ductal carcinoma may merit further investigation with larger cohorts and multimodal analyses. Highlights • Epstein-Barr virus DNA was detected in 4.6% of invasive breast cancer tissues. • No Human Herpesvirus 8, BK polyomavirus, or JC polyomavirus DNA was found in any breast tissue samples. • Epstein-Barr encoded RNA transcripts were not detected in DNA-positive samples using in situ hybridization. • Findings suggest limited involvement of Epstein-Barr virus in breast cancer pathogenesis. • Combined molecular and histopathological approaches are recommended for future studies.