Aims: To examine bacterial contamination of passenger aircraft and to identify aeroplane environments posing the greatest potential health risk. Methods and Results: DNA was extracted from ten environmental samples collected on four different flights (three domestic, one international) from a variety of surfaces frequently touched by passengers. PCR clone libraries were made from the DNA samples using bacterial-specific 16S ribosomal DNA (rDNA) primers. A total of 271 bacterial rDNA sequences were obtained. We used BLAST analysis of the rDNA clone sequences to identify sequences in Genbank with the highest sequence similarity. The majority of the rDNA clones obtained from aeroplane environments were more than 97% identical to rDNA sequences from cultured bacterial species. Samples collected from the cabin surfaces (e.g., tray tables and arm rests) had undetectable levels of DNA and produced no PCR products. Bacterial diversity was highest on lavatory surfaces, including door handles, toilet handles, and sink faucets. Sequence data from these surfaces detected species from 58 different bacterial genera, and many of the best BLAST hits matched rDNA sequences of cultured species known to be opportunistic pathogens. The most frequently observed species came from five genera commonly associated with humans: Streptococcus, Staphylococcus, Cornybacterium, Proprionibacterium and Kocuria. Conclusions: The results show that there is a large diversity of bacterial contamination on aeroplanes, including organisms known to be opportunistic pathogens. Significance and Impact of the Study: Our results indicate that aeroplanes have the potential to spread an enormous diversity of bacterial species among passengers and destinations. Aeroplane lavatories present an especially significant concern to public health.