Bovine parainfluenza virus type 3 (BPIV-3) and Bovine alphaherpesvirus-1 (BoHV-1) lead to severe diseases in domesticated animals, such as Bovine, sheep, and goats. One of these diseases is mastitis, whose signs may not be observable in cases of viral infection due to the dominance of other clinical symptoms. This may lead to failure to predict viral agents in subclinical Bovine cases. Since viral infections have not been substantially investigated in mastitis studies, information about immune response to BPIV-3 and BoHV-1 infected Bovine mammary tissues may be inadequate. The present study aimed to determine the presence and prevalence of BPIV-3 and BoHV-1 agents in Bovine mammary tissues, and the immune response of such tissues against BPIV-3 and BoHV-1 infection. For this purpose, we first detected these viruses with qRT-PCR in mammary tissues. Then, we determined the expression profiles of interferon-γ (IFN-γ), CD4, and CD8 genes with qRT-PCR. Lastly, we performed immunohistochemistry staining to identify the presence of IFN-γ, CD4, and CD8 proteins in the mammary tissues. We found that 26, 16, and five of the 120 samples were BPI3-, BoHV1-, and BPIV-3 + BoHV-1 infected, respectively. Moreover, the gene expression levels of IFN-γ and CD4 were strongly up-regulated in the virus-infected tissues, whereas the CD8 gene expression level was only moderately up-regulated. Immunohistochemistry staining results were consistent with qRT-PCR results. Overall, our findings showed a high prevalence of BPIV-3 and BoHV-1 and indicated that cell-mediated immune response plays an important role against BPIV-3 and BoHV-1 infection in Bovine mammary tissues. Meanwhile, IFN-γ is an important cytokine for antiviral immunity against such infection.