Cupriavidus metallidurans CH34 and Pseudomonas putida mt2 were used as cadmium (Cd) resistant and sensitive bacteria, respectively to study the effect of Cd on physicochemical surface properties which include the study of surface charge and hydrophobicity which are subjected to vary under stress conditions. In this research work, effective concentration 50 (EC₅₀) was calculated to exclude the doubt that dead cells were also responding and used as reference point to study the changes in cell surface properties in the presence of Cd. EC₅₀ of C. metallidurans CH34 was found to be 2.5 and 0.25 mM for P. putida mt2. The zeta potential analysis showed that CH34 cells were slightly less unstable than mt2 cells as CH34 cells exhibited −8.5 mV more negative potential than mt2 cells in the presence of Cd in growth medium. Cd made P. putida mt2 surface to behave as intermediate hydrophilic (θ_w = 25.32°) while C. metallidurans CH34 as hydrophobic (θ_w = 57.26°) at their respective EC₅₀. Although belonging to the same gram-negative group, both bacteria behaved differently in terms of changes in membrane fluidity. Expression of trans fatty acids was observed in mt2 strain (0.45%) but not in CH34 strain (0%). Similarly, cyclopropane fatty acids were observed more in mt2 strain (0.06–0.14%) but less in CH34 strain (0.01–0.02%). Degree of saturation of fatty acids decreased in P. putida mt2 (36.8–33.75%) while increased in C. metallidurans CH34 (35.6–39.3%). Homeoviscous adaptation is a survival strategy in harsh environments which includes expression of trans fatty acids and cyclo fatty acids in addition to altered degree of saturation. Different bacteria show different approaches to homeoviscous adaptation.