Here, we aimed at the preparation of an antibacterial surface on a flexible polydimethylsiloxane substrate. The polydimethylsiloxane surface was sputtered with silver, deposited with carbon, heat treated and exposed to excimer laser, and the combinations of these steps were studied. Our main aim was to find the combination of techniques applicable both against Gram-positive and Gram-negative bacteria. The surface morphology of the structures was determined by atomic force microscopy and scanning electron microscopy. Changes in surface chemistry were conducted by application of X-ray photoelectron spectroscopy and energy dispersive spectroscopy. The changes in surface wettability were characterized by surface free energy determination. The heat treatment was also applied to selected samples to study the influence of the process on layer stability and formation of PDMS-Ag or PDMS-C-Ag composite layer. Plasmon resonance effect was determined for as-sputtered and heat-treated Ag on polydimethylsiloxane. The heating of such structures may induce formation of a pattern with a surface plasmon resonance effect, which may also significantly affect the antibacterial activity. We have implemented sputtering of the carbon base layer in combination with excimer laser exposure of PDMS/C/Ag to modify its properties. We have confirmed that deposition of primary carbon layer on PDMS, followed by sputtering of silver combined with subsequent heat treatment and activation of such surface with excimer laser, led to the formation of a surface with strong antibacterial properties against two bacterial strains of S. epidermidis and E. coli.