Objectives The aims of this study were to investigate the antimicrobial efficacy of antiseptics in saliva-derived microcosm biofilms, and to examine phenotypic adaption of bacteria upon repeated exposure to sub-inhibitory antiseptic concentrations. Methods Saliva-derived biofilms were formed mimicking caries- or gingivitis-associated conditions, respectively. Microbial compositions were analyzed by semiconductor-based 16S rRNA sequencing. Biofilms were treated with CHX, CPC, BAC, ALX, and DQC for 1 or 10 min, and colony forming units (CFU) were evaluated. Phenotypic adaptation of six selected bacterial reference strains toward CHX, CPC, and BAC was assessed by measuring minimum inhibitory concentrations (MICs) over 10 passages of sub-inhibitory exposure. Protein expression profiles were investigated by SDS-PAGE. Results Both biofilms showed outgrowth of streptococci andVeillonellaspp., while gingivitis biofilms also showed increased relative abundances ofActinomyces,Granulicatella, andGemellaspp. Antiseptic treatment for 1 min led to no relevant CFU-reductions despite for CPC. When treated for 10 min, CPC was most effective followed by BAC, ALX, CHX, and DQC. Stable adaptations with up to fourfold MIC increases were found inE. colitoward all tested antiseptics, inE. faecalistoward CHX and BAC, and inS. aureustoward CPC. AdaptedE. colistrains showed different protein expression as compared with the wildtype strain. Conclusion Antiseptics showed limited antimicrobial efficacy toward mature biofilms when applied for clinically relevant treatment periods. Bacteria showed phenotypic adaptation upon repeated sub-inhibitory exposure.