Tuning surface composition and stiffness is now an established strategy to improve the integration of medical implants. Recent evidence suggests that matrix stiffness affects bacterial adhesion, but contradictory findings have been reported in the literature. Distinguishing between the effects of bacterial adhesion and attachment strength on these surfaces may help interpret these findings. Here, we develop a precision microfluidic shear assay to quantify bacterial adhesion strength on stiffness-tunable and biomolecule-coated silicone materials. We demonstrate that bacteria are more strongly attached to soft silicones, compared to stiff silicones; as determined by retention against increasing shear flows. Interestingly, this effect is reduced when the surface is coated with matrix biomolecules. These results demonstrate that bacteria do sense and respond to stiffness of the surrounding environment and that precisely defined assays are needed to understand the interplay among surface mechanics, composition, and bacterial binding.