Bacterial adhesion and colonization are complicated processes that depend on many factors, including surface chemistry, hydrophobicity, and surface roughness. The contribution of each of these factors has not been fully elucidated because most previous studies used different polymeric surfaces to achieve differences in properties. The objective of this study was to modify hydrophobicity and roughness on one polymeric surface, eliminating the confounding contribution of surface chemistry. Mechanically assembled monolayer (MAM) preparation methods (both one- and two-dimensional) were used to impart different degrees of hydrophobicity on fluoroalkylsilane (FAS)-coated silicone. Surface roughness was varied by casting the silicone to templates prepared with different abrasives. Surface hydrophobicity was determined by contact angle measurement, whereas surface roughness was determined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Bacterial adhesion and colonization were analyzed using a direct colony-counting method and SEM images. Hydrophobicity increased as a function of stretched length or width (Deltax or Deltay); it reached a maximum at Deltax = 60% with one-dimensional MAM and decreased as Deltax further increased to 80 and 100%. The same trend was observed for the two-dimensional MAM. After 12-h incubation, all the FAS/silicone surfaces had significantly reduced adherence of Staphylococcus epidermidis by 42-89%, compared to untreated silicone, and the degree of which is inversely related to surface hydrophobicity. On the other hand, surface roughness had a significant effect on bacterial adhesion and colonization only when the root-mean-square roughness was higher than 200 nm.