In this paper, we present an investigation into the effects of surface topography (roughness) and hydrophobicity (contact angle measurement) on bacteria adhesion for polystyrene materials. The surfaces of polystyrene substrates were patterned using UV-laser radiation with a wavelength of 193 nm under different conditions. Various surface topographies were fabricated and were measured by an optical surface profiler and contact angle measurements were recorded. For the bacterial adhesion experiments, an assay of Escherichia coli (E. coli) was developed and used for measurements on both as-received and modified polystyrene surfaces. The method is based on the staining of attached bacterial cells with the nucleic acid-binding, green fluorescent DAPI (4′, 6-diamidino-2-phenylindole) stain. The preliminary results show that laser-assisted modification induced by laser ablation can make polystyrene substrates either more hydrophilic (with oxygen) or more hydrophobic (with air). The contact angle can be varied from 37° to 108°. The results on bacterial attachment show that the polystyrene substrates as received have no bacteria attached, indicating a good anti-bacterial performance. The treated substrates show some bacterial attachment and, in particular, the surfaces with a high contact angle have much higher numbers of bacterial cells attached. This indicates that such a laser-assisted process with air can make polystyrene surfaces more attractive to E. coli bacteria.