The aim of the study was to identify Pseudoalteromonas strain OS-9 and evaluate its anti-Vibrio potential. Based on the morphological, biochemical, and 16S rRNA sequence analysis, the strain was identified as Pseudoalteromonas haloplanktis. Experiments designed with Box Behnken showed that cell density and cell-free supernatant activity were simultaneously maximum at 30 degrees C, pH 7, and 90 h. The coefficient values obtained from the response surface methodology regression equations determined that pH was more effective on responses than other physical variables. The cell-free supernatant of P. haloplanktis OS-9 extracted under optimized conditions was only effective on Gram-negative fish pathogenic bacteria and especially on Vibrio. Among Vibrio spp., the strongest inhibitory effect was observed against V. rotiferianus. At the maximum cell density (10.48 OD), the strain OS-9 showed the maximum inhibitory effect against V. rotiferianus (26.42 mm zone diameter). In coculture assay, the growth of V. rotiferianus was inhibited after 96 h with an initial level of 1.0 x 10(6) CFU ml(-1) by the strain OS-9. These results indicated that P. haloplanktis strain OS-9 could be considered as a potential Vibrio bio-controlling agent.