Diabetic foot ulcers (DFUs) are complex polymicrobial infections with high risk of chronicity, amputation, and mortality. Understanding microbial dynamics and adaptive mechanisms is critical for improving therapeutic strategies. In this study, we performed longitudinal microbiome profiling of DFU samples (n=30) to investigate microbial composition associated with wound outcomes- healed (n=10), intermediate (n=10), and worsened (n=10), along with non-DFU samples (n=15). Pseudomonas aeruginosa was consistently detected across all cohorts, highlighting its central role in DFU microbiota. Other clinically relevant pathogens, including Staphylococcus aureus, Acinetobacter baumannii, and Enterococcus spp., were also identified, with differential abundance patterns linked to wound progression. Functional predictions indicated enrichment of pathways associated with biofilm formation, virulence, and antimicrobial resistance in worsened wounds. To further investigate adaptive resistance mechanisms, adaptive laboratory evolution (ALE) was performed on ATCC strains of these four key pathogens under antibiotic pressure. Whole genome sequencing of evolved populations revealed mutations in genes associated with DNA repair (rec, mut, uvr), cell envelope regulation (walK, vraF), translation machinery (ribosomal proteins and rRNA modification enzymes), and global regulatory systems (agrA, codY). These genetic changes align with functional pathways predicted from microbiome analysis, suggesting convergence between community-level functional potential and strain-level adaptive evolution. Overall, this study integrates longitudinal microbiome profiling with experimental evolution to demonstrate that key DFU pathogens undergo coordinated genetic adaptation in pathways linked to virulence, persistence, and antimicrobial resistance. These findings provide mechanistic insights into DFU pathogenesis and highlight potential targets for therapeutic intervention.