Extracellular DNA in municipal wastewater and effluents from hospitals and R&D laboratories contains antimicrobial resistance and recombinant genes that are today considered as a new class of emerging contaminants. The objective of this research was to investigate the effect of disinfection agents on the integrity of DNA molecules by using real-time PCR. Escherichia coli cell suspensions and genomic DNA in aqueous solution were exposed to increasing doses of disinfection systems, including chlorination, UV irradiation, silver ions, and TiO2 nanoparticles/near-UV. The doses resulting in damage of DNA (16S rDNA) were determined using real-time PCR and compared with the doses resulting in the inactivation of bacterial cells. Our results showed that the disinfection agents chlorine, UV, and silver significantly inhibited the amplification of a fragment of 16S rDNA, but only when applied at doses much higher than the lethal doses for E. coli bacteria. The inactivation doses of TiO2 nanoparticles/near-UV were of the same order of magnitude for both DNA and living cells. Our results raise questions about the efficacy of disinfection processes to destroy and prevent the dispersion of DNA pollutants into the environment. In addition, the damage of DNA by high levels of disinfectants may have implications for the utilization of PCR-based methods for bacterial detection.