Background The advent and use of highly sensitive molecular biology techniques to explore the microbiota and microbiome in environmental and tissue samples have detected the presence of contaminating microbial DNA within reagents. These microbial DNA contaminants may distort taxonomic distributions and relative frequencies in microbial datasets, as well as contribute to erroneous interpretations and identifications. Results We herein report on the occurrence of bacterial DNA contamination within commonly used DNA extraction kits and PCR reagents and the effect of these contaminates on data interpretation. When compared to previous reports, we identified an additional 88 bacterial genera as potential contaminants of molecular biology grade reagents, bringing the total number of known contaminating microbes to 181 genera. Many of the contaminants detected are considered normal inhabitants of the human gastrointestinal tract and the environment and are often indistinguishable from those genuinely present in the sample. Conclusions Laboratories working on bacterial populations need to define contaminants present in all extraction kits and reagents used in the processing of DNA. Any unusual and/or unexpected findings need to be viewed as possible contamination as opposed to unique findings. Electronic supplementary material The online version of this article (doi:10.1186/s13099-016-0103-7) contains supplementary material, which is available to authorized users.