The extraction method and particularly the type of solvents used in the procedure are one of the most crucial steps for isolating antimicrobial compounds and extracts that potentially affect various microorganisms. This study aimed to evaluate the effects of water, ethanol (100%), methanol (100%), ethanol-water (50:50), and methanol-water (50:50) burdock (Arctiumlappa) root extracts (BE) on total phenol content and polyphenols as well as the antioxidant activity. In the next step, the solvent with the highest efficiency to have phenolic compounds were selected and employed to investigate the antibacterial effects of BE on the pathogenic bacterial strains (i.e. Yersinia ruckeri, Lactococcus garvieae, Pseudomonas putida and Aeromonas hydrophila) in rainbow trout (Oncorhynchus mykiss) by using disc diffusion test and tube dilution techniques. Maximum mean total phenolic content and antioxidant capacity were detected in the water-methanol mixture. Additionally, High-performance liquid chromatography(HPLC) analysis revealed that both hesperidin and trans-ferulic acid compounds existed in all solvents. The antimicrobial activity of water-methanol BE was varied based on the type of microorganism. In this context, Y. ruckeri as a gram negative bacterium was found to be the most susceptible one compared to other pathogens. According to our findings, water-methanol solvent was more efficient to isolate phenolic compounds and exert antioxidant capacity, as the level of phenolic compounds in the extract was increased. Moreover, Y. ruckeri was recognized as the most sensitive pathogen in all tests.