Streptococcus mitis
is a commensal bacterial species of the oral cavity, with the potential for opportunistic pathogenesis. For successful colonization,
S. mitis
must be able to adhere to surfaces of the oral cavity and survive and adapt to frequently changing environmental conditions. Cyclic‐di‐AMP (c‐di‐AMP) is a nucleotide second messenger, involved in the regulation of stress responses and biofilm formation in several bacterial species. Cyclic‐di‐AMP is produced by diadenylate cyclases and degraded by phosphodiesterases. We have previously shown that in
S. mitis
, one diadenylate cyclase (CdaA) and at least two phosphodiesterases (Pde1 and Pde2) regulate the intracellular concentration of c‐di‐AMP. In this study, we utilized
S. mitis
deletion mutants of
cdaA
,
pde1
, and
pde2
to analyze the role of c‐di‐AMP signaling in various stress responses, biofilm formation, and adhesion to eukaryotic cells. Here, we demonstrate that the Δ
pde1
mutant displayed a tendency toward increased susceptibility to acetic acid at pH 4.0. Deletion of
cdaA
increases auto‐aggregation of
S. mitis
but reduces biofilm formation on an abiotic surface. These phenotypes are more pronounced under acidic extracellular conditions. Inactivation of
pde1
or
pde2
reduced the tolerance to ciprofloxacin, and UV radiation and the Δ
pde1
mutant was more susceptible to Triton X‐100, indicating a role for c‐di‐AMP signaling in responses to DNA damage and cell membrane perturbation. Finally, the Δ
pde2
mutant displayed a tendency toward a reduced ability to adhere to oral keratinocytes. Taken together, our results indicate an important role for c‐di‐AMP signaling in cellular processes important for colonization of the mouth.