Probiotic bacteria have been suggested to inhibit Streptococcus mutans (SM) and thus prevent dental caries. However, supporting evidence is weak and probiotic species might be cariogenic themselves. Thus, we compared and combined the probiotic Lactobacillus rhamnosus GG (LGG) with SM and analysed the resulting mineral loss (ΔZ) in dental tissues. We simulated three biofilm compositions (SM, LGG, SM × LGG), two lesion sites (smooth enamel, dentin cavity) and two nutrition supply frequencies (twice/day, 6 times/day) in a multi-station, continuous-culture biofilm model. A total of 240 bovine enamel and dentin samples were cut, polished and embedded. All experimental procedures were performed in independent duplicates, with 10 samples being allocated to each group for each experiment (final sample size n = 20/group). Biofilms were cultured on the specimens and supplied with 2% sucrose medium and artificial saliva in consecutive pulses. After 10 days, ΔZ and bacterial numbers were assessed. SM × LGG biofilms caused significantly increased ΔZ compared with SM or LGG biofilms (p < 0.01, Mann-Whitney test), and ΔZ was significantly increased in dentin cavities compared with smooth enamel lesions (p < 0.01). Bacterial numbers did not significantly differ between biofilms of different species (p > 0.05, ANOVA). Frequent nutrition supply significantly increased bacterial numbers (p < 0.01). Biofilms in dentin cavities compared to smooth enamel harboured significantly more bacteria (p < 0.05). LGG induced mineral loss especially in dentin cavities and under highly cariogenic conditions. LGG did not have inhibitory effects on SM, but rather contributed to the caries process in vitro.