3,3'-Diaminobenzidine (DAB) is a widely used chromogen in histological staining methods and stained tissue is often used in downstream molecular analyses such as quantitative PCR (qPCR). Using microdissected muscle fibers from sequential muscle sections stained by DAB-dependent and -independent methods, we show that DAB exerts a strong inhibitory effect on qPCR-based mitochondrial DNA quantification. This effect introduces a significant bias in the estimation of mitochondrial DNA copy number and deletion levels between DAB-positive and -negative fibers. We reproduce our findings in microdissected neurons from human brain tissue, suggesting a general effect of DAB staining on PCR analyses independent of the underlying tissue or cell type. Using an exogenous DNA template added to tissue samples we provide evidence that DAB-staining predominantly interferes with the tissue-derived DNA template rather than inhibiting DNA polymerase activity. Our results suggest that DAB-based staining is incompatible with PCR-based quantification methods and some of the previously reported results employing this approach should be reconsidered.