Background: The role of carcinogenic viruses in developing breast cancer has not yet been identified. Many studies have examined the association between breast cancer and human cytomegalovirus (HCMV), but conflicting results have been reported. Objectives: The aim of this study was to evaluate the levels of IgM & IgG antibodies against HCMV by identifying the viral genome in the breast tissue of women with breast cancer. Methods: A total of 60 patients with breast cancer and 60 healthy individuals (40 cases with fibroadenoma and 20 healthy samples) were selected. Serum levels of IgM & IgG antibodies against HCMV were measured by ELISA, and after DNA extraction from the breast tissue, the presence of the cytomegaloviruses (CMV) genome was assessed by Real-Time PCR. Results: Real-time PCR results showed that 20 samples of breast cancer tissue and 5 samples of fibroadenoma were positive for CMV genome (P = 0.001, OR: 5.50, CI 95%: 1.90 - 15.89). All samples had CMV-IgG antibody in their serum, but their mean serum level was higher in the cancer group (48.27 ± 15.99 U/mL) than the control group (40.11 ± 18.01 U/mL) (P = 0.004). However, CMV-IgM anti-viral antibody was positive in 5 cases with cancer and 3 cases in the control group. The mean serum concentration of this antibody was higher in the cancer group (6.60 ± 6.75 U/mL) than the control group (4.92 ± 3.03 U/mL) (P = 0.099). Conclusions: Increased serum levels of anti-CMV antibodies in patients with cancer, as well as the presence of viral genomes in some cases, indicate the carcinogenesis effect of the virus.