Antimicrobial agents are now being studied in bacterial biofilms rather than in suspensions. However, no data are available whether the choice of substratum influences biofilm properties or the assessment of antimicrobial efficacy. This study compared dentin and polyacrylate (PA) grooves as substratum for biofilm formation on pH profiles after sugar challenge, as well as biofilm susceptibility to chlorhexidine. Streptococcus mutans biofilms were formed in a constant depth film fermenter (CDFF) by alternate pulsing with sucrose solutions and bacterial growth medium. Spatial and temporal pH profiles were measured using a pH microelectrode outside the CDFF in a flow reactor. Also, 15-d biofilms were treated with either water or 0.2% chlorhexidine and tested for total viable counts and organic acid content. The results show that in PA grooves, compared with dentin grooves, minimum pH was lower but recovered faster after a sucrose pulse. The pH response to sugar application decreased with the age of the biofilm more in PA than in dentin grooves. Chlorhexidine treatment in PA grooves gave greater reductions in viable counts and acid formation than in dentin. We conclude that the nature of the substratum not only affects metabolic activity of biofilms but also their susceptibility to antimicrobials.