16S ribosomal RNA gene sequences are characteristically used as gold-standard genetic marker for the determination of bacterial and/or archaeal biodiversity, and community profiling of environmental samples. The 16S rRNA amplicon analysis till-date is taken as a standard method for investigation and identification of uncultivable bacteria in microbial diversity studies. The accuracy of these analyses strongly depends upon the choice of primers. It is presumed that these primers do not participate in non-specific amplifications. In the present study, by in silico, PCR and denaturing gradient gel electrophoresis (DGGE) analysis, we have shown that primers do cross-react with eukaryotic DNAs as well, eventually leading to overestimation of microbial biodiversity. We further demonstrated that the overestimation is not only due to cross-reaction with eukaryotic mitochondrial or plastid DNA, but also with eukaryotic chromosomal DNA, that is ubiquitous in environmental samples. We tried to establish methanogenic diversity in municipal solid waste (MSW) leachates and cow dung samples before and after enrichment of the prokaryotic DNA from eukaryotic ones. Results revealed that bands disappeared/get lightened in bacterial 16S rRNA-based DGGE community profiles, after prokaryotic DNA enrichment, but not in mcrA-based community profiles.