Aquilaria agallocha is an economically valuable plant facing endangerment, sought after globally for its production of agarwood. A. agallocha tree possesses medicinal and aromatic properties in its fruits, branches, leaves, wood, and roots, which are being studied for their effective compounds and their potential bioactive effects on human health. This study aimed to uncover the phytochemical properties and biological activities of A. agallocha roots, which possess medicinal and aromatic characteristics. The phenolic, organic acid, and sugar profiles of A. agallocha roots were determined using HPLC-DAD, while mineral distributions were analyzed via ICP-AES. In vitro antioxidant capacity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Furthermore, the antibacterial effects of methanol extracts of A. agalocha roots against S. aureus, S. carnosus, B. cereus, L. monocytogenes, E. coli, L. innocua, K. pneumoniae, and E. faecalis were estimated with disc diffusion and MIC methods. The amount of total phenolic and in vitro DPPH radical scavenging capacity of A. agalocha roots were determined as 7.529 mg GAE/g and 18.83 µmol TE/g, respectively. Eight phenolic components were detected in the extracts of A. agalocha roots. The most domi nant among these phenol i c compounds detected was tannic acid, which was followed by 4-hydrobenzoic acid and oleuropein, respectively. In the aqueous extracts of A. agalocha roots, four organic acids were identified, including citric, tartaric, malic and succinic acid, and succinic acid was the most dominant organic acid. Only fructose sugar was detected in the water extracts of A. agalocha roots. The extracts of A. agalocha roots had a strong antibacterial effect against all gram-positive pathogens except for S. carnosus, but did not have any effect against gram-negative bacteria.