Vibriosis, caused by Vibrio parahaemolyticus, is a major bacterial disease in shrimp aquaculture, often managed with antibiotics that contribute to antimicrobial resistance and environmental concerns. This study investigated the antimicrobial properties of Kiam wood (Cotylelobium lanceotatum) extract and its potential as a dietary supplement to enhance the disease resistance of Pacific white shrimp, Penaeus vannamei. Kiam wood extracts were prepared using ethanol-water mixture at different ratios, and their antimicrobial activity was evaluated against V. parahaemolyticus. The water extract (KWE) exhibited the strongest anti-Vibrio activity as indicated by the widest clearance zone (15.65 mm), with a minimum inhibitory concentration (MIC) of 256 µg/mL and a minimum bactericidal concentration (MBC) of 512 µg/mL. Further assays demonstrated that KWE effectively inhibited biofilm formation and restricted bacterial motility at 512 µg/mL (MBC). Scanning electron microscopic images revealed significant cell-wall damages in treated bacteria as shown by membrane disruption and pore formations. Liquid chromatography-mass spectrometry (LC-MS) analysis identified Amuresins D (CHO), Pauciflorol A (CHO), Vaticasides A (CHO), Vaticanols B (CHO), and Cotylelophenol B (CHO) as key bioactive compounds. For the in vivo trials, P. vannamei was firstly infected by feeding them a V. parahaemolyticus-impregnated diet (1.5 × 10⁴ CFU/g) for two days, followed by a 15-day feeding period with KWE-supplemented diets at 2MBC (1 mg/g). Shrimp-fed KWE diets showed a significant reduction in intestinal Vibrio loads, enhanced immune responses (total hemocyte count, semi-granulocyte, and granulocyte levels), and improved survival rates. Notably, the KWE diet achieved the highest survival rate (85%), compared to 52% in the control group. These findings highlight KWE as a promising natural antimicrobial agent with immunostimulatory properties, offering a sustainable alternative to synthetic antibiotics for managing vibriosis in shrimp aquaculture. Further studies should explore its mode of action and long-term effects on shrimp health and aquaculture environments.