Ethyl caffeate (EC) is a naturally occurring compound found in Bidens pilosa with a range of health-promoting effects, such as antibacterial activity and antioxidant properties. Hybrid groupers have rapid growth rates, strong disease resistance, and short production cycles. However, nowadays, the intensification of hybrid grouper farming has led to poor water quality, with the main bacterial disease being Vibrio sp. This study evaluated the effect of EC on the antimicrobial activity against Vibrio sp., the nonspecific immune responses in vitro and in vivo, as well as the challenge test against Vibrio parahaemolyticus. The EC concentration range of 5–50 µg/mL was able to inhibit the growth of V. parahaemolyticus, V. alginolyticus, and V. harveyi with values of 19.7 ± 0.56, 19.3 ± 1.53, and 20.6 ± 1.53 mm. Moreover, The EC can potentially increase the metabolite seconder activity of beneficial bacteria; for instance, when combined with LAB and P. acidilactici, it can completely kill the Vibrio sp. at 6 hours incubation time. The EC has been proven to have no toxic effect based on MTT assay in head kidney leukocytes at a concentration of 0.5-10 µg/mL and has antioxidant activity that is able to enhance ROS production and phagocytic activity. At the transcriptional level, EC supplementation can upregulate the activity of TOR, NF-kB, and Nrf2 signaling pathway-related genes. The EC supplementation of the diet increased the immune activity on the systemic and mucosal organs after post-challenge with V. parahaemolyticus, resulting in a higher survival rate. Further research is needed to determine whether EC supplementation can support growth performance.