AbstractGracilaria verrucosa seaweed is a type of seaweed commonly found in water. This study was conducted to investigate the effect of G. verrucosa on fish pathogenic bacteria to support fish farming. The method used in this research was the separation of G. verrucosa fractions using column chromatography. The active antibacterial fraction of G. verrucosa which is obtained from column chromatography indicated fractions containing antibacterial compounds. It was fraction number 3 by using an eluent 16 (ethanol): 4 (ethyl acetate). Furthermore, based on phytochemical screening, ultraviolet spectrophotometer and LC–MS analysis, antibacterial compounds contained in those fraction number 3 are Alkaloid, Flavonoid, Tannin, Phenolic compound. Based on LC–MS and UV–Vis analysis, flavonoid group, Quercetin-7-methyl-ether is a dominant group of the antibacterial compound on fraction no. 3. This fraction had moderate antibacterial activity against Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas putida and had weak antibacterial activity against Vibrio harveyi and Vibrio algynoliticus bacteria.