Background and objectives: Though dental caries has a multifactorial etiology, the specific factor that results in its high rate of transmissibility is the microbial component, that is, mutans streptococci (MS), and its association with protective factors, such as salivary immunoglobulin A (S-IgA). Hence, it is important to identify such risk and protective factors associated with early childhood caries (ECC) using isolation of the colonies based on the fundamental method of colony morphology, adjunct with an advanced fully automated platform, to minimize human errors. Materials and methods: A retrospective analysis was done on 40 children who were divided into two groups: group I (experimental) and group II (control). Plaque samples from the children were collected by scraping the material from four index teeth using a sterile toothpick. The samples were transported to the central research laboratory for estimation of viable MS colonies on selective media TYCSB (tryptone, yeast extract, cysteine w/ sucrose, w/ bacitracin) and the VITEK® 2 COMPACT automated microbial identification system. The data obtained were subjected to statistical analysis. Results: On comparison of viable MS between varying severities of dental caries and caries-free children in the age-group 3–6 years, it was found to be below the grand median of 33 colony-forming unit (CFU) × 10⁴ for subgroup IA and control group II. A significant positive statistical correlation (r = 0.960) was present between the viable MS and varying severities of ECC and the control group. Group IB had a significant relationship for both the variables (MS colony count and S-IgA) among all other subgroups and the control group as well. Interpretation and conclusion: Multiple comparison of mean difference in MS CFU and S-IgA levels between different severities of dental caries using Tukey's HSD range test was established.