This study investigated the spatial distribution patterns of airborne bacterial concentrations and microbial community structures in a modern commercial layer facility housing approximately 50,000 laying hens equipped with advanced environmental control systems. Air samples were systematically collected at 50 strategically distributed locations using a six-stage Andersen microbial air sampler, while environmental samples (dust, manure, intestinal contents) were characterized using 16S rRNA gene sequencing. Results demonstrated a distinct longitudinal gradient in airborne bacterial concentrations, progressively increasing from the air inlet (883±177 CFU/m³) to exhaust fans (12,650±813 CFU/m³), with a facility-wide mean concentration of 5,618±530 CFU/m³. Spatial analysis revealed significant bacterial concentration heterogeneity, with elevated bacterial loads (>
8,000 CFU/m³) concentrated in central regions while peripheral areas maintained lower concentrations (<
6,000 CFU/m³). Taxonomic profiling identified Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes as predominant phyla across all sample types, with significant compartment-specific distribution patterns: Firmicutes dominated intestinal samples (72.9 %), Proteobacteria and Bacteroidetes were enriched in posterior dust and manure samples, while Acinetobacter exhibited highest abundance (19.90 %) in anterior dust. Differential abundance analysis demonstrated significant enrichment of fecal-associated bacteria (particularly Bacteroides and Escherichia coli) in posterior sampling locations, establishing direct correlations between environmental parameters and microbial dissemination patterns. This comprehensive spatial-microbial analysis elucidates critical factors influencing bacterial dispersion within intensive poultry production environments, providing the empirical foundation for implementing concentration-based risk stratification management systems and targeted interventions to enhance biosecurity, minimize disease transmission, and optimize poultry health in commercial operations.