Purpose: The aim of this study was to investigate the influence of oral implant surface roughness on bacterial biofilm formation and antimicrobial treatment efficacy. Materials and Methods: Titanium disks with low-roughness pickled surfaces and with moderately rough sandblasted, acid-etched surfaces were used as substrata. Streptococcus mutans biofilms (1 and 3 days old) and Porphyromonas gingivalis biofilms (3 days old) were grown on the two types of substrata and then treated with 0.2% chlorhexidine. Biofilm viability was evaluated by a resazurin metabolism assay and by sonication-colony-forming unit counts. Results: Surface roughness had no influence on the amount of biofilm formation by S mutans or P gingivalis in this in vitro biofilm model. However, it strongly affected the treatment efficacy of chlorhexidine on the biofilms formed by both species. Higher roughness resulted in lower efficacy. Furthermore, treatment efficacy was significantly reduced in older biofilms. Conclusion: A moderately roughened surface did not enhance biofilm formation but reduced treatment efficacy of the biofilms. This finding indicates that efforts should be directed toward optimizing implant surface properties for effective antimicrobial treatment without compromising osseointegration.