Micron-scale and submicron-scale surface roughness enhance osteoblast differentiation on titanium (Ti) substrates and increases bone-to-implant contact in vivo. However, bacterial adhesion is also strongly influenced by roughness and surface chemistry. The aim of this study was to investigate if chemical surface modifications alter initial bacterial attachment. To achieve this, two polyelectrolyte layers [chitosan (Ch) and poly(L-lysine) (PLL)] were used to coat Ti surfaces with different roughness (PT [Ra<0.3μm], SLA [Ra≥3.0μm]). Bacterial attachment was evaluated using Aggregatibacter actinomycetemcomitans, Actinomyces israelii, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum, Parvimonas micra, Porphyromonas gingivalis, Prevotella intermedia and Streptococcus sanguinis. After 24h incubation, bacteria were detached from the samples with sonication and the counting plate technique was performed to determine the number of colony forming units (CFU's). Additionally, surfaces were observed by scanning electron microscopy to determine bacteria surface coverage. Statistical significance was determined using ANOVA followed by Bonferroni's modification of Student's t-test. The results showed that polyelectrolyte coatings did not affect surface roughness. Modified surfaces were more hydrophilic than the controls. PT surfaces covered by Chi exhibited lower CFUs than the same surface covered by PLL or the control PT (140 × 105/mL, 343 × 105/mL and 283 × 105/mL, respectively). The opposite effect was observed on the SLA surfaces, PLL coated samples shown lower CFUʼs than Chi or uncoated SLA (199 × 105/mL, 229 × 105/mL and 227 × 105/mL, respectively). The Chi layer appeared to reduce bacterial adhesion only on the smooth surfaces. In contrast, PLL coatings reduced bacterial attachment on rougher surfaces. These results suggest that chemical modification of Ti without alteration of surface roughness affects oral bacterial attachment, and could be useful to prevent peri-implantitis related diseases.