Arachidonic acid (AA) exhibits anti-bacterial and anti-biofilm activities against Streptococcus mutans, a cariogenic bacterium responsible for dental caries. The study investigated the effects of AA on S. mutans, revealing that it has both bactericidal and bacteriostatic effects. The minimum inhibitory concentration (MIC) of AA was 25 µg/ml in the presence of 5% CO₂ and reduced to 6.25–12.5 µg/ml in its absence. The anti-bacterial activity was accompanied by changes in membrane properties, including increased membrane fluidity, membrane perforation, and altered membrane transport. AA also showed anti-biofilm effects, with the minimum biofilm inhibitory concentration (MBIC) matching the MIC, suggesting that part of the anti-biofilm effect was due to the anti-bacterial activity. Gene expression studies indicated that AA reduced the expression of biofilm-related genes, suggesting a direct anti-biofilm effect. Flow cytometric analyses using SYTO 9/PI staining, fluorescent efflux pump substrates, and DAPI staining showed that AA leads to membrane hyperpolarization, altered membrane transport, and increased membrane permeability. AA also acts as an antioxidant, with its anti-bacterial activity being abrogated by the lipid peroxyl radical scavenger α-tocopherol. AA was found to be non-toxic to normal Vero epithelial cells and did not cause hemolysis of erythrocytes. These findings suggest that AA is a potentially safe drug that can be used to reduce the bacterial burden of cariogenic S. mutans. The study highlights the potential of AA as a novel therapeutic agent for the prevention of dental caries.Arachidonic acid (AA) exhibits anti-bacterial and anti-biofilm activities against Streptococcus mutans, a cariogenic bacterium responsible for dental caries. The study investigated the effects of AA on S. mutans, revealing that it has both bactericidal and bacteriostatic effects. The minimum inhibitory concentration (MIC) of AA was 25 µg/ml in the presence of 5% CO₂ and reduced to 6.25–12.5 µg/ml in its absence. The anti-bacterial activity was accompanied by changes in membrane properties, including increased membrane fluidity, membrane perforation, and altered membrane transport. AA also showed anti-biofilm effects, with the minimum biofilm inhibitory concentration (MBIC) matching the MIC, suggesting that part of the anti-biofilm effect was due to the anti-bacterial activity. Gene expression studies indicated that AA reduced the expression of biofilm-related genes, suggesting a direct anti-biofilm effect. Flow cytometric analyses using SYTO 9/PI staining, fluorescent efflux pump substrates, and DAPI staining showed that AA leads to membrane hyperpolarization, altered membrane transport, and increased membrane permeability. AA also acts as an antioxidant, with its anti-bacterial activity being abrogated by the lipid peroxyl radical scavenger α-tocopherol. AA was found to be non-toxic to normal Vero epithelial cells and did not cause hemolysis of erythrocytes. These findings suggest that AA is a potentially safe drug that can be used to reduce the bacterial burden of cariogenic S. mutans. The study highlights the potential of AA as a novel therapeutic agent for the prevention of dental caries.