Reusable water bottles used in daily life are prone to surface scratches caused by repeated cleaning and scrubbing. Such scratches promote bacterial adhesion and biofilm formation (Lomander et al., 2004). Accordingly, this study aimed to determine an optimal cleaning frequency that effectively removes bacterial proliferation in reusable water bottles, the objective was to identify an appropriate cleaning interval. Considering practical usability, Tritan copolyester exhibited superior performance in inhibiting bacterial retention. Using a standardized cleaning protocol, four cleaning frequencies were established: no cleaning, daily cleaning, once every three days, and once every seven days. Samples were collected from both the inner surface and the water content of the bottles for bacterial culture. The results indicated that regular cleaning significantly reduced microbial residues, whereas extended cleaning intervals led to a rapid increase in bacterial colonies. Furthermore, Gram-positive bacteria, suggesting a stronger capacity for proliferation in humid environments. Therefore, it is recommended that Tritan copolyester bottles be cleaned at consistent intervals and thoroughly dried after washing to effectively prevent bacterial regrowth and ensure drinking water hygiene and safety.