Introduction: Maintaining good oral hygiene is an important factor in health.Toothbrushes are commonly used to maintain oral health and prevent dental disease, but unfortunately how keeping the toothbrush is neglected . The aim of this study was to investigate the relationship between toothbrush keeping method and its microbial content.

Methods: In this cross-sectional study, 60 volunteers were enrolled and divided into 3 groups based on the places of keeping their toothbrushes (bedroom, bathroom and lavatory). The participants were asked to brush once a day for one month using the first toothbrush which had been delivered then the first toothbrushes were gathered and a second toothbrush was delivered. The participants were asked to brush once a day using the second toothbrush for 3 months. All toothbrushes were sent for culture and evaluation. All toothbrushes were evaluated by a blind microbiologist. Toothbrush bristles were washed in BHI broth medium then the resulting liquid was cultured in MacConkey’s agar for gram-negative bacteria and in blood agar and chocolate agar for gram-positive bacteria. Colony counts of Streptococcus mutans, Candida albicans, Pseudomonas, Klebsiella, S. aureus, and E. coli were determined and multiplied by one thousand. Data were analyzed by SPSS version 18 and using Kruskal-Wallis test.

Results: At the end of the study the results showed statistically significant differences in microbial load between the groups (p=0.014). Toothbrushes that were kept in bathroom had highest microbial load.

Conclusions: Toothbrushes kept in the bathroom had the greatest microbial contamination after three months. According to the results of this study, bathroom is the worst place and bedroom is the best place for keeping toothbrushes.