Objective. The aim of this study was to develop in vitro biofilms on SLA titanium (Ti-SLA) and zirconium oxide (ZrO2) surfaces and to evaluate the effect of antiseptic agents on the number of putative periodontal pathogenic species. Methods. An in vitro biofilm model was developed on sterile discs of Ti-SLA and ZrO2. Three antiseptic agents [chlorhexidine and cetyl-pyridinium-chloride (CHX/CPC), essential oils (EEOOs) and cetyl-peridinium-chloride (CPC)] were applied to 72-h biofilms, immersing discs during 1 min in the antiseptic solution, either with or without mechanical disruption. Viable bacteria [colony forming units (CFU/mL)] were measured by quantitative polymerase chain reaction (qPCR) combined with propidium monoazide. A generalized lineal model was constructed to determine the effect of the agents on the viable bacterial counts of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum on each surface. Results. The exposure to each antiseptic solution resulted in a statistically significant reductions in the number of viable target species included in the in vitro multi-species biofilm, on both Ti-SLA and ZrO2 (p < 0.001) which was of up to 2 orders for A. actinomycetemcomitans, for P. gingivalis 2 orders on Ti-SLA and up to 3 orders on ZrO2, and, for F. nucleatum up to 4 orders. No significant differences were found in counts of the tested bacteria between in vitro biofilms formed on both Ti-SLA and ZrO2, after topically exposure to the antimicrobial agents whether the application was purely chemical or combined with mechanical disruption. Significance. A. actinomycetemcomitans, P. gingivalis and F. nucleatum responded similarly to their exposure to antiseptics when grown in multispecies biofilms on titanium and zirconium surfaces, in spite of the described structural differences between these bacterial communities. (C) 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.