The aim of this study was to evaluate the promotive and/or inhibitory effects of NaCl, glucose, their combinations on biofilm formation and quorum sensing (QS) autoinducer-2 (AI-2) production on black tiger shrimp surfaces, using three strains of Vibrio parahaemolyticus. Initially, six different NaCl concentrations (0.5, 1, 2, 3, 4, and 5%) were evaluated for a maximum biofilm formation (∼6.3 log CFU/cm2) at 30 °C. Inhibitory effects of glucose at six different levels (0.005, 0.01, 0.015, 0.02, 0.025, and 0.05%) were then investigated using the NaCl level previously found for a maximal biofilm formation. V. parahaemolyticus formed the best biofilm at 2% NaCl and the least biofilm at 5% NaCl, regardless of vibrio strain. In combination of 2% NaCl and glucose at different levels, the largest biofilm was observed at 0.015%, with the least biofilm seen at 0.05%, regardless of vibrio strain. Addition of salt and glucose more than the optimal level (2% salt and 0.015% glucose at 2% salt) inducted a stepwise inhibition of vibrio growth and biofilm formation in a continuous matter. In the visual evaluation, similar results were observed for vibrio growth, biofilm formation, live/dead cell detection, and quorum sensing.