In this study, Escherichia coli RP437 and Pseudomonas aeruginosa PAO1 were used as model strains to investigate the early stage biofilm formation on poly(dimethylsiloxane) (PDMS) surfaces with varying stiffness, which were prepared by controlling the degree of cross-linking (base:curing agent ratios of 5:1, 10:1, 20:1, and 40:1 were tested). An inverse correlation between cell adhesion and substrate stiffness was observed for both species. Interestingly, it was found that the cells attached on relatively stiff substrates (5:1 PDMS) were significantly smaller than those on relatively soft substrates (40:1 PDMS). In addition to the difference in size, the cells on 5:1 PDMS substrates were also found to be less susceptible to antibiotics, such as ofloxacin, ampicillin, and tobramycin, than the cells attached on 40:1 PDMS substrates. These results reveal that surface stiffness is an important material property that influences the attachment, growth, and stress tolerance of biofilm cells.