Purpose: To evaluate the use of hydrogen peroxide as an adjunct to ultrasonication (US) in biofilm removal and whether it can limit the spread of viable microorganisms in the aerosol. Materials and Methods: Multi-species biofilms were formed on dentin disks and titanium disks fixed on a plastic surface. After placing the specimens in a periodontal pocket model, an ultrasonic scaler was applied for 30 s, in part combined with 0.25% or 0.5% H2O2. After treatment, the remaining biofilm was analysed for bacterial counts (colony forming units [CFU]), biofilm quantity and metabolic activity. Further, the cytotoxic effect of hydrogen peroxide on periodontal ligament fibroblasts was assessed and the spread of bacteria in aerosol was quantified. Results: Ultrasonication reduced bacterial counts in biofilm, biofilm mass and metabolic activity on both dentin and titanium disks. Adjunctive use of 0.25% and 0.5% H2O2 more effectively reduced the viable bacteria in biofilm than ultrasonication alone; this was also found on both dentin and titanium. The different concentrations of H2O2 did not lead to corresponding differences in bacterial mass and metabolic activity. The spread of bacteria through aerosols was statistically significantly reduced when adjunctive H2O2 was used. However, a certain cytotoxic effect on periodontal ligament fibroblasts by H2O2 could not be ruled out. Conclusions: Irrigating with H2O2 during periodontal instrumentation with an ultrasonic scaler increases the reduction of viable bacteria within biofilms. It might limit bacterial spreading via aerosols.