Background: Streptococcus mutans and Lactobacilli play an important role in the etiopathogenesis and progression of dental caries (DC). Their quantification and identification may be helpful for epidemiological and early intervention measures. Objectives: We conducted the study to evaluate the colony counts of S. mutans and Lactobacillus with the location of DC and correlate their prevalence with the age of the patient. Materials and methods: The study population comprised 60 patients with DC. They were divided into two groups according to age, and each group was further divided into three subgroups based on involvement of enamel, dentin, and pulp by DC. The swab samples were collected, and organisms were isolated using Mitis Salivarius Bacitracin (MSB) Agar and Lactobacillus MRS Agar. Manual counting of colonies on plates illuminated by transmitted light was done. Results were summarized and analyzed statistically. Results: The caries prevalence was found to be higher in children, with females being more affected. In both groups, posterior teeth were more affected, and occlusal/incisal surface caries were more common. The mean colony count of S. mutans (61.3%) and Lactobacillus (63.4%) was significantly higher in group I compared to group II. In both groups, the mean colony counts of S. mutans were higher in enamel, followed by dentin and pulp. In contrast, in both groups, the mean colony counts of Lactobacillus were higher in pulp, followed by dentin and enamel. Conclusion: Bacterial colony counts may help in taking specific measures against specific organisms and thereby prevent the development of new carious lesions.