Halophilic actinobacteria produce secondary metabolites with diverse biological activities. This study investigates bioactive compounds from termite mound isolates collected across Virudhunagar, Tamil Nadu. Twenty halophilic isolates were screened; only ten tolerated 12% NaCl. Among them, isolate N3 exhibited strong antibacterial activity against Klebsiella pneumoniae MTCC 39 and Staphylococcus aureus MTCC 96, and antifungal activity against Candida albicans MTCC 282. Bioactive metabolites were purified using TLC and identified via GC-MS, revealing diketopiperazine- and peptide-based compounds with antimicrobial, antitumor, and anti-inflammatory potential. 16S rRNA sequencing confirmed isolate N3 as Microbacterium barkeri (98% similarity). The study highlights the potential of halophilic actinobacteria as sources of novel antimicrobial agents and proposes possible coupling pathways between hydroxyl and carbonyl groups within bioactive diketopiperazines.