Introduction: Stingless bee acts as a pollinator and is commonly found in tropical dry and humid forest. Various phenolic compounds have been reported in stingless bee honey which gave antibacterial, antifungal, and antiviral properties. Objective: The current study focuses on the chemical profiling of honey from Heterotrigona itama on Borneo and evaluation for potential therapeutic properties. Methods: The honey was extracted via liquid-liquid extraction method and analyzed using spectroscopic methods. Results: Strong fatty alcohol signals (3.5-4.5 ppm) indicated significant markers in 1H-NMR. The honey demonstrates excellent antibacterial activity against Escherichia coli (E. coli) (19.0 mm) and stronger antioxidant properties (IC50 33.78 ppm) compared to the Tualang honey (54.90 ppm). Bornean H. itama honey produced lower glucose (61.65-147.44 g/L) and heavy metals content (0.003-0.204 ppm) which is significant in food products. Conclusion: The phenolic, aliphatic acids and fatty alcohols identified in honey contributing to excellent biological properties. This study demonstrated that Borneo H. itama honey is a potential source of antimicrobial and antioxidant agents.