Surface stiffness plays a critical role in bacterial adhesion, but the mechanism is unclear since the bacterial motion before adhesion is overlooked. Herein, the three-dimensional (3D) motions of Escherichia coli and Pseudonomas sp. nov 776 onto poly(dimethylsiloxane) (PDMS) surfaces with varying stiffness before adhering were monitored by digital holographic microscopy (DHM). As Young's modulus (E) of the PDMS surface decreases from 278.1 to 3.4 MPa, the adhered E. coli and Pseudonomas sp. decrease in number by 40.4 and 34.9%, respectively. Atomic force microscopy (AFM) measurements show that the adhesion force of bacteria to the surface declines with the decreased surface stiffness. In contrast, a nontumbling mutant of adhered E. coli (HCB1414 with the adaptive function being partially deficient) decreases much less (by 18.4%). On the other hand, the tumble frequency (F-t) of E. coli HCB1 and flick frequency (F-f) of Pseudomonas sp. increase as the surface stiffness decreases, and the motion bias (B-theta) of Pseudomonas sp. also increases. These facts clearly indicate that the bacteria have adapted responses to the surface stiffness. RNA sequencing (RNA-seq) reveals that the downregulated Cph2 and CsrA as well as the upregulated GcvA of swimming E. coli HCB1 in bulk near the softer surface promote the bacterial motility.