<p>This study employs a comprehensive multi-omics approach to investigate the regulatory roles of specific microRNAs (miRNAs) in Congenital Tooth Agenesis (CTA). A total of 58 miRNAs associated with tooth diseases, cancer, and bone development were initially identified through a literature review and analyzed using bioinformatics. Based on target prediction and network analysis, eight miRNAs with strong connectivity and common target genes were shortlisted for further investigation. Blood samples from 10 CTA patients and 5 healthy controls were analyzed for miRNA expression using stem-loop RT-PCR. Four miRNAs—hsa-miR-218-5p, hsa-miR-15b-5p, hsa-miR-200b-3p, and hsa-let-7a-3p—were identified as significantly differentially expressed, marking their first reported involvement in CTA. Notably, hsa-miR-218-5p and hsa-let-7a-3p emerged as novel regulators with no prior associations with CTA or tooth development. To address the limitations of a small sample size, a multi-omics strategy was employed to validate these findings, integrating miRNA expression data with whole exome sequencing (WES), gene expression panels, and metabolomic profiling. The analysis confirmed the association of these four miRNAs with CTA and highlighted their involvement in critical biological pathways such as Wnt signaling, FGF signaling, and PI3 kinase pathways, which are essential for cellular proliferation, differentiation, and tissue morphogenesis. Importantly, the identification of these miRNAs in blood samples, rather than traditional dental tissues, highlights a minimally invasive approach that could aid in the early detection, therapeutic targeting, and personalized management of dental anomalies.</p> Graphical Abstract <p></p>