Persistence of the antivibrio property of the potential antagonistic probiotics, Pseudomonas MCCB 102 and 103, at different temperatures, pH and in organic solvents was studied. The antivibrio compound was extracted, purified and characterized using thin-layer chromatography, high-pressure liquid chromatography, liquid chromatography-mass spectroscopy, UV-Vis and nuclear magnetic resonance spectroscopy and identified as N-methyl-1-hydroxyphenazine, a phenazine antibiotic. The toxicity of the compound was tested in Penaeus monodon haemocyte culture and the IC50 value was found to be 1.4 ± 0.31 mg L-1. The compound was found to be bacteriostatic at 0.5 mg L-1. Its stability to varying temperature, pH, organic solvents, prolonged shelf-life and vibriostatic nature point to its suitability for prophylatic aquaculture application. © 2009 The Authors. Aquaculture Research © 2009 Blackwell Publishing Ltd.