BACKGROUND AND AIM: The most common oral hygiene aid used for the maintenance of oral health is the toothbrush and its hygiene is frequently neglected. Cigarette smoking is associated with many oral and periodontal diseases. Its effect can never be neglected on the microbiota of the oral cavity. Therefore, this study was planned to evaluate the microbial contamination of toothbrushes and its relation with smoking. This study aimed to assess the microbial contamination of capped and uncapped toothbrushes of adult smoker and non-smoker men. METHODS: This randomized study included 50 different used toothbrushes of healthy men, non-smoker and smoker, who may use capped and uncapped toothbrushes. This study was conducted at Karachi Medical and Dental College, Karachi, Pakistan, from September to October 2019. All toothbrushes were evaluated for microbial analysis. Samples were allocated through simple random sampling. Every toothbrush head was put in a sterile container with 10 ml brain heart agar (Oxoid) and incubated in 37 º, C for 24 hours,after which, they were placed in different bases (blood agar, McConkey). Results were analyzed using SPSS software. RESULTS: Staphylococcus aureus was the most offender and major organism causing contamination in all 4 groups of smokers and non-smokers with capped toothbrush and smokers and non-smokers with uncapped toothbrush. A correlation test was done using Pearson’, s correlation, showing a positive correlation between uncapped non-smokers and capped smokers (P = 0. 050). CONCLUSION: The microbial contamination of capped and uncapped toothbrushes of adult smoker and non-smoker men was quite high and hence, it increases the risk of dental diseases.