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<b>Aim:</b> To study and compare the microbial flora of dental plaque after the use of a self-contaminated toothbrush and that of a single-use toothbrush. <b> Materials and Methods:</b> The study group included 40 young volunteers from Yenepoya Dental College, who were free from any systemic or oral disease. In these subjects, plaque samples were collected after 1 month use of a self-contaminated toothbrush. Each subject was given a set of 30 new toothbrushes and a toothpaste tube and instructed to use one toothbrush everyday and discard it after use. The plaque samples were collected on a weekly interval and cultured on Mitis Salivarius agar. The colonies were identified and speciated and their count was recorded. <b> Results:</b> Streptococcus mitis, S. mutans, S. sanguis, S. milleri and Candida were recovered from the samples. A highly significant decrease in their numbers was found after the use of a single-use toothbrush (P value 0.001). <b>Conclusions</b> : As a contaminated toothbrush can reintroduce microorganisms into the oral cavity, it may be a sound practice to change the toothbrush as frequently as possible.