Two pairs of genes were identified in Streptococcus mutans with similarity to relBE and mazEF toxin-antitoxin (TA) modules of Escherichia coli. Transcription of mazEF and relBE was repressed by amino acid starvation, and relBE expression was repressed by low pH. Mutants lacking MazF, RelE, or both toxins (MRT1) grew in broth media and formed biofilms as well as the parent. Biofilm populations of MRT1 were more resistant to acid killing than the parent or single mutants. MRT1 also exhibited a longer diauxie during growth on glucose and inulin and displayed decreased phosphoenolpyruvate:sugar phosphotransferase activity. This is the first report that demonstrates a physiological role for TA modules in Gram-positive bacteria.