Introduction: Fluid flow has a prominent influence on the metabolism of surface-attached biofilms. Dental biofilms are covered by a thin saliva film that flows at different rates in different locations under stimulated and unstimulated conditions. Methods:The present study employed pH ratiometry to study the impact of different flow velocities, saliva film thicknesses and saliva concentrations on microscale pH developments in Streptococcus mutans biofilms of different age. Results:While saliva flow at a velocity of 0.8 mm/min (unstimulated flow) had little impact on biofilm pH, stimulated flow (8 mm/min; 80 mm/min) affected vertical pH gradients in the biofilms and raised the average pH in 48-h biofilms, but not in 72-h and 168-h biofilms. The saliva film thickness had a strong impact on biofilm pH under both static and dynamic conditions. pH drops were significantly higher in biofilms exposed to a thin saliva film (≤ 50 µm) than a thick saliva film (> 50 µm). pH drops in the biofilms were also strongly dependent on the saliva concentration and thus the buffer capacity of the salivary medium. For 48-h and 72-h biofilms, but not for 168-h biofilms, pH drops in distinct microenvironments were more pronounced when the local biofilm thickness was high.