The long-term influence of silicone oil 200 cSt (SO200) and 2, 2, 4, 4, 6, 8, 8-heptamethylnonane (HMN) on the cell surface hydrophobicity (CSH) of a hexane-degrading Pseudomonas aeruginosa strain and a toluene-degrading Pseudomonas putida strain was assessed in two-phase partitioning bioreactors under batch and continuous operation. CSH was evaluated using a modified BATH method based on optical density (CSH(OD)) and colony-forming unit (CSH(CFU)) measurements. In the presence of HMN, P. aeruginosa turned hydrophobic over the time course as shown by the gradual increase in CSH(OD) (61 ± 1%) and CSH(CFU) (53 ± 3%) under batch degradation and in CSH(OD) (49 ± 0%) under continuous operation. However, P. putida turned hydrophobic only under continuous operation ([Formula: see text]). On the other hand, no significant CSH enhancement was observed in both Pseudomonas strains in the presence of SO200. These results suggested that CSH is species, non-aqueous phase, and cultivation mode dependant, and an inducible property of bacteria. Maximum hexane elimination capacities increased by 2 and 3 in the presence of SO200 and HMN, respectively. Based on the absence of CSH in P. aeruginosa in the presence of SO200, the higher elimination capacities recorded were likely due to an improved hexane mass transfer (physical effect). However, in the presence of HMN, a direct hexane uptake from the non-aqueous phase (biological effect) might have also contributed to this enhancement.