The purpose of this study was to evaluate the effect of seawater (SW) on the activity of antibiotics belonging to 10 families (aminoglycosides, aminopenicillins, carbapenems, β-lactams, chloramphenicols, monobactams, nitrofurans, quinolones, sulfonamides, and tetracyclines) against Vibrio strains isolated from hemolymph of Pacific white shrimp, Litopenaeus vannamei, farmed in Northeastern Brazil and standard strain Vibrio cholera ATCC 19582. Susceptibility of the strains to antibiotics was determined by disk diffusion method and the minimum inhibitory concentration was determined by macrodilution method. The media Mueller–Hinton agar and broth used in the above methods were diluted in distilled water (control, 1% NaCl, pH 7.5) and SW (2.5% NaCl, pH 7.5). The antibiotics most affected by dilution in SW were tetracycline, penicillin, cephalothin, aztreonam, ampicillin, and imipenem, as indicated by a considerable increase in the number of strains classified as intermediate or resistance. Thus, in this study, the efficiency of these antibiotics on Vibrio strains was found to be reduced by contact with SW.