studies show that DNase can inhibit and biofilm formation. However, the underlying molecular mechanisms remain poorly understood. This study used an RNA-sequencing transcriptomic approach to investigate the mechanism by which DNase I inhibits early and biofilm formation on a transcriptional level, respectively. A total of 1171 differentially expressed genes (DEGs) in and 1016 DEGs in enriched in a variety of biological processes and pathways were identified, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs were primarily involved in two-component system, biofilm formation, and flagellar assembly and in biosynthesis of secondary metabolites, microbial metabolism in diverse environments, and biosynthesis of amino acids, respectively. The transcriptional data were validated using quantitative real-time polymerase chain reaction (RT-qPCR), and the expression profiles of 22 major genes remained consistent. These findings suggested that DNase I may inhibit early biofilm formation by downregulating the expression of genes associated with flagellar assembly and the type VI secretion system, and by downregulating capsular polysaccharide and amino acids metabolism gene expression, respectively. This study offers insights into the mechanisms of DNase treatment-based inhibition of early and biofilm formation.