Oral diseases (e.g., dental caries, periodontitis) are developed when the healthy oral microbiome is imbalanced allowing the increase of pathobiont strains. Common practice to prevent or treat such diseases is the use of antiseptics, like chlorhexidine. However, the impact of these antiseptics on the composition and metabolic activity of the oral microbiome is poorly addressed. Using two types of oral biofilms—a 14-species community (more controllable) and human tongue microbiota (more representative)—the impact of short-term chlorhexidine exposure was explored in-depth. In both models, oral biofilms treated with chlorhexidine exhibited a pattern of inactivation (>3 log units) and fast regrowth to the initial bacterial concentrations. Moreover, the chlorhexidine treatment induced profound shifts in microbiota composition and metabolic activity. In some cases, disease associated traits were increased (such as higher abundance of pathobiont strains or shift in high lactate production). Our results highlight the need for alternative treatments that selectively target the disease-associated bacteria in the biofilm without targeting the commensal microorganisms.