Abstract  Background:  Root caries, increasingly prevalent in aging populations, poses a significant challenge for oral health. Gingival recession exposes tooth roots, leading to vulnerability to microbial colonization. However, the microbial factors influencing the progression of root caries in Bangladesh have not been extensively studied. Aim:  The aim of this study was to evaluate the prevalence of Streptococcus mutans , Lactobacillus , and Actinomyces  species in root carious lesions in middle-aged and older adults in the Bangladeshi population and to comparatively assess the microbial count between the two age groups. Materials and Methods : A total of 180 adult patients aged 35-75 years were included in the study, with 90 individuals in the middle-aged group (35-44 years) and 90 in the older group (55-75 years). Specimens from root carious lesions were collected for bacteriological analysis using standard culture techniques. Isolation and identification of Streptococcus mutans , Lactobacillus , and Actinomyces  species were performed on selective media. Results:  The microbial analysis was performed using the Chi-square test with SPSS 25. Streptococcus mutans  was identified in 56.7% of samples, Lactobacillus  in 28.3%, and Actinomyces  species in 15%. These microorganisms were predominantly found in the older age group, with a significant association between the age group and the presence of Lactobacillus  (P=0.024). Conclusions : This study found that both aerobic Gram-positive cocci ( Streptococcus mutans , Lactobacillus ) and anaerobic bacteria ( Actinomyces  spp.) were more prevalent in root carious lesions in older adults compared to middle-aged individuals in Bangladesh. The findings underscore the age-related microbial shifts in root caries and suggest the need for targeted prevention and treatment strategies for the aging population.