The influence of mechanical stiffness of biomaterials on bacterial adhesion is only sparsely studied and the mechanism behind this influence remains unclear. Here, bacterial adhesion on polydimethylsiloxane (PDMS) samples, having four different degrees of stiffness with Young's modulus ranging from 0.06 to 4.52 MPa, is investigated. Escherichia coli and Pseudomonas aeruginosa are found to adhere in greater numbers on soft PDMS (7- and 27-fold increase, respectively) than on stiff PDMS, whereas Staphylococcus aureus adheres in similar numbers on the four tested surfaces. To determine whether the observed adhesion behavior is caused by bacteria-specific mechanisms, abiotic polystyrene (PS) beads are employed as bacteria substitutes. Carboxylate-modified PS (PS-COOH) beads exhibit the same adhesion pattern as E. coli and P. aeruginosa with four times more adhered beads on soft PDMS than on stiff PDMS. In contrast, amine-modified PS (PS-NH2 ) beads adhere in similar numbers on all tested samples, reminiscent of S. aureus adhesion. This work demonstrates for the first time that the intrinsic physicochemical properties associated with PDMS substrates of different stiffness strongly influence bacterial adhesion and challenge the previously reported theory on active bacterial mechanosensing, which provides new insights into the design of antifouling surfaces.