The most common method of oral hygiene is tooth brushing. However, occasionally brushing becomes a risk factor for health. The main reason is getting infected with various microorganisms. The purpose of this study was to investigate bacterial and fungal contamination of toothbrushes students, and factors affecting the contamination.
 Materials & Methods: Ninety five brushes were assessed in this study. Samples were incubated in test tubes containing nutrient broth for twenty-four hours. Then the microorganisms were isolated using specific nutrient medium containing Blood agar, Chocolate agar MacConkey and sabouraud dextrose agar. The inoculated plates were incubated for 24 to 48 hours at 37 C. The identification of micro-organisms was performed based on biochemical tests.
 Findings: The infection rate was more than 40% of the subject staphylococcus, Niesseria, Diphteroids,Actinomycetes and Enterobacteriacae family members were isolated from samples. Microbial contamination was higher in women than men (P<0.05).There was found correlation between the number of microorganism on brush with time of use, using mouthwash and gender (P<0.05). There was statistically significant relation between cocci microorganisms’ infections with sexes as well as between bacillus infections with capped brush.
Discussion & Conclusion: Based on results, high incidence of bacterial contamination observed in brushes, therefore the proper use, maintenance and timely replacement of toothbrush play an important role in reducing pollution and consequently decreasing oral disease.