Research on naturally produced new antimicrobial agents is of great importance for discovering novel pharmaceutical sources. This study aimed to investigate the antibacterial properties of four different concentrations of the ethanolic extract of a premix of brown macroalgae (Padinaaustralis, Sargassum ilicifolium, and Stoechospermum marginatum) and standard antibiotics (Gentamicin (10 µg), Ciprofloxacin (5 µg), and Chloramphenicol (30 µg)) against pathogenic bacteria (Vibrio parahemolyticus, V. harveyi, V. alginolyticus, and acute hepatopancreatic necrosis disease (AHPND+)) as well as the probiotic bacterium Lactobacillus plantarum in marine aquatic species.After sampling and transferring the samples to the laboratory, the brown macroalgae samples P. australis, S. ilicifolium, and S. marginatum were washed, dried, mixed in equal ratios (1:1:1:1), and ground. Extraction was performed using ethanol solvent. The ethanol extract was prepared at four concentrations of 10, 20, 40, and 80 mg/mL to investigate the antibacterial effect of the aqueous extract of the macroalgae premix on four Gram-negative bacterial species (V. parahemolyticus, V. harveyi, V. alginolyticus, and AHPND+) and the probiotic bacterium L. plantarum using the agar disk diffusion test. The results showed that different concentrations of the ethanol extract of the brown macroalgae premix had no inhibitory effect on the growth of the Gram-positive bacterium L. plantarum and the Gram-negative V. alginolyticus, with no inhibition zones observed. Additionally, the bacteria V. parahemolyticus, V. harveyi, and AHPND+ showed less sensitivity to various concentrations of the ethanol extract of the brown macroalgae premix compared to the standard antibiotics tested (P>0.05). The effect of the ethanol extract of the macroalgae premix at different concentrations on the AHPND+ bacterium did not show a significant difference (P<0.05). The bacterium V. harveyi showed the least sensitivity to the ethanolic extract of permix, with a growth inhibition zone diameter of 3.20 ± 0.20 mm at a concentration of 80 mg/mL. This value was significantly different from the standard antibiotics gentamicin, ciprofloxacin, florfenicol, and other concentrations (P > 0.05). The gram-negative bacterium V. parahemolyticus did not show significant differences at concentrations of 10, 20, and 40 mg/mL (P < 0.05). Numerically, the highest sensitivity of V. parahemolyticus was observed at a concentration of 10 mg/mL, which was significantly different from the 80 mg/mL concentration of the ethanolic permix extract. Overall, although the antibacterial property of the aqueous extract of the studied brown macroalgae permix was calculated to be lower than that of standard antibiotics, the brown macroalgae permix extract can be introduced as a potential source of novel natural antimicrobial compounds and, considering their abundance, can be utilized for pharmaceutical and dietary supplement applications.