The study aimed to determine whether the red fluorescence (RF) of a dental microcosm biofilm as measured with quantitative light-induced fluorescence (QLF) technology is useful for assessing the efficacy of antimicrobials. Dental microcosm biofilms were formed on bovine enamel discs and grown under 0.3% sucrose challenge and treated with chlorhexidine (CHX) solutions at different concentrations (0.05%, 0.1%, and 0.5%) plus a negative control [sterile distilled water (DW)] twice daily for 7 days. The biofilms were photographed using a QLF-digital system to evaluate the RF by calculating the red/green ratio, and pH values of the medium were measured daily. After 7 days, the bacterial viability of the biofilm was assessed by measuring the counts of viable total bacteria and aciduric bacteria, and the percentage surface microhardness changes (%SHC) was evaluated. The RF and cariogenic properties were compared for the different concentrations of CHX, and their correlations were examined. The RF and its increase rate were much lower for CHX-treated biofilms than for DW-treated biofilms. The RF after 7 days of maturation decreased significantly with increasing CHX concentrations (
) and was from 31% (for 0.05% CHX) to 46% (for 0.5% CHX) lower than that of the DW group. Strong correlations were reported between the RF of the 7-day-maturation biofilms and cariogenic properties, such as the number of total bacteria (
), number of aciduric bacteria (
), supernatant pH (
), and %SHC (
). In conclusion, the RF of dental biofilms as measured with QLF technology can be used to nondestructively assess and monitor the effect of antimicrobials against biofilm.