This study evaluated the antibacterial effects of short-time exposure of surface pre-reacted glass-ionomer (S-PRG) eluate on oral microcosm biofilm. Biofilms were treated with an S-PRG eluate at different concentrations (25%, 50%, and 100%), distilled water (DW), and 0.1% chlorhexidine (CHX) twice a day for 5 min repeatedly. After 7 days, the total and aciduric bacterial counts and biofilm dry weights were measured. An image analysis program calculated the red/green (R/G) ratios in the biofilm autofluorescence images. Microscopic analyses quantified the biofilm thickness and live/dead cell ratio and determined morphological changes in the biofilm. Bacterial counts and dry weights were not significantly different in the DW group for all S-PRG eluate concentrations. An increasing trend in the R/G ratio for 7 days biofilm treatment was observed for the S-PRG eluate and the DW groups. Furthermore, the live/dead cell ratios in the biofilm and the biofilm thickness of the S-PRG eluate groups were similar to those of the DW group. The bacteria morphology inside the biofilm changed only in the CHX group. Short-time S-PRG eluate treatment showed no significant antibacterial and antibiofilm effects. These results indicated that limited biofilm formation inhibition can be obtained by using only the S-PRG eluate.