BACKGROUND This study aimed to evaluate the relationship of the level of salivary peptides human neutrophil defensin (HNP) 1-3 in children with and without early childhood caries (ECC). METHODS This in vitro study was conducted among 86 children of age 3-6 years who were divided into two groups: Group 1 - children with ECC (n = 43) and Group 2 - children without ECC (n = 43). Saliva samples were collected, and salivary peptide HNP1-3 levels were analyzed using enzyme-linked immunosorbent assay. The data collected were subjected to appropriate statistical analysis. Independent sample t-test was used to compare the mean salivary peptide levels of HNP1-3 in children with and without ECC. One-way ANOVA was used for intragroup comparison of the mean peptide levels between the ages. P < 0.05 was considered statistically significant. RESULTS The mean age of the children in Group 1 and Group 2 was 5.12 ± 0.851 and 4.88 ± 0.879 years, respectively. A statistically significant decrease was seen in salivary peptide HNP1-3 levels in children with ECC (1.44 ng/ml) when compared to children without ECC (6.04 ng/ml) with P < 0.001. There were no statistically significant differences in the gender- and age-based comparisons. CONCLUSIONS A decrease in salivary peptide HNP1-3 levels might be a biological factor for predisposition to ECC and hence can be used as a predictive and a preventive tool in caries prevention.