Novel approaches for the prevention of biofilm-mediated oral diseases aim to control dental biofilms rather than eradicating bacteria in the mouth. One such approach is the use of enzymes that specifically target and degrade the dental biofilm matrix and thereby facilitate biofilm removal. Matrix-degrading enzymes have consistently shown promising results , but data on -grown oral biofilms are limited. This study aimed to investigate the effect of combined treatment with mutanase, beta-glucanase and DNase on biofilm formation and removal, microbial biofilm composition and biofilm pH. Biofilms from healthy participants were grown for 48 or 72 h on lower-jaw splints and enzyme or control-treated during (3x/day, 30 min) or after growth (30 min). Under the tested conditions, enzyme treatment had no significant effect on biofilm formation or removal compared to control, as assessed by optical coherence tomography and confocal microscopy. Likewise, enzymatic treatment did not induce significant changes in the microbial composition of the biofilms that were dominated by , , , and species. The biofilm pH response to a sucrose challenge was assessed using confocal microscopy-based pH ratiometry, and the average biofilm pH was not significantly different between the intervention groups. Under the conditions employed in this study, the tested enzymes had no significant impact on grown biofilms. The treatment regimen, the biofilm composition, or the analytical methods employed may explain the difference to previous results. Further studies are warranted to assess the therapeutic potential of multi-enzyme treatment for dental biofilm control.