Reusable water bottles are growing in popularity, but consumers regularly refill bottles without a corresponding effort at cleaning them. If the difficulties associated with various bottle designs and materials are added in, it is clear that improperly cleaned water bottles may present a potential contamination risk and thus be a risk for foodborne illness. The purpose of this study was to measure contamination levels of water bottles that are in use and to investigate bottle usage and cleaning behaviors by collecting survey data from the bottle owners. Total organic materials on the exterior surface and coliform and heterotrophic bacteria on the interior surface were enumerated, using ATP bioluminescence and the agar plate count method, respectively. The HPC and coliform results revealed a marked microbial contamination level among reusable water bottles that are in use, and the ATP levels suggest that the exterior bottle surfaces may serve as fomites that facilitate the transmission of infectious organisms. The contamination level can be affected by factors such as bottle material, refill frequency, beverage type, and cleaning behavior.