Neonatal meconium represents a relatively underexplored microbial source that may harbour microorganisms with probiotic potential, uniquely adapted for intestinal colonization and metabolic health. From 120 meconium samples, 167 spore-forming isolates were screened to identify strains exhibiting desirable probiotic functional and safety attributes. Three strains (ZBOE6, ZBRV8, and ZBTM2) were selected and identified as Bacillus subtilis through whole-genome sequencing. These strains showed non-haemolytic (α/γ types) and DNase negative phenotypes, indicating non-pathogenic profile, along with detectable bile salt hydrolase (Bsh) activity. The isolates exhibited tolerance to adverse stressors, including acidic conditions, bile salts, and simulated gastric and intestinal conditions. Isolates showed epithelial adhesion potential (up to 87.5% for ZBRV8) and auto-aggregation efficiencies ranging from 60 to 80%. In vitro assays using Caco-2 cell lines demonstrated anti-adhesive potential against E. coli through competitive, exclusion, and displacement mechanisms. The strains showed cholesterol assimilation potential greater than 60% and antioxidant activity up to 62.7% (DPPH scavenging) and 97.9% (hydroxyl radical scavenging). In vitro safety assessment revealed no biogenic amine production, minimal cytotoxicity (<30%) in the Vero cell line, and susceptibility to clinically relevant antibiotics with minor strain-dependent variation consistent with intrinsic resistance profiles. Enzyme assay profiling revealed activity for lipase, phytase, amylase, and β-galactosidase. Genomic analysis further supported these phenotypic observations by identifying genes associated with stress tolerance, adhesion, metabolism, and antimicrobial activity, providing preliminary mechanistic insight into strain-specific functional traits. Overall, these findings suggest that Bacillus subtilis isolated from human neonatal meconium represent promising probiotic candidates; however, their functional efficacy and safety require further validation through in vivo and clinical studies for gut and metabolic health interventions.