Bacterial predilection for organic solvents is important in whole-cell biocatalysis in organic media. Although various methods of measuring bacterial hydrophobicity have been proposed, it is not fully determined whether they are applicable to the assessment of bacterial predilection for organic solvents in whole-cell biocatalytic processes. In this study, bacterial predilection for organic solvents was assessed by bacterial adhesion to hydrocarbon (BATH), contact angle measurement (CAM), hydrophobic interaction chromatography (HIC), and glass adhesion test (GAT). These methods were applied to the cultures of four bacterial species of industrial importance, namely, Rhodococcus opacus B-4, R. erythropolis PR4, Pseudomonas putida T-57, and Escherichia coli JM109, in organic media. Experimental results revealed that CAM assays could be used to predict the dispersibility of bacterial cells in anhydrous organic solvents. However, when bacteria were suspended in aqueous-organic (A/O) two-phase media, the results of BATH assays provided the most reliable assessment of bacterial predilection for organic solvents. This discrepancy noted between CAM and BATH assays was attributed to the effect of electrostatic interaction between bacteria and oil droplets. In A/O two-phase media, the accessibility of a water-immiscible dye, nile red, to the bacterial cell surface, correlated well with BATH assay results.