The study investigates the anti-bacterial and anti-biofilm activities of arachidonic acid (AA) against *Streptococcus mutans*, a cariogenic bacterium. AA, a polyunsaturated fatty acid, was found to have a minimum inhibitory concentration (MIC) of 25 μg/ml in the presence of 5% CO2, reducing to 6.25–12.5 μg/ml without CO2. The anti-bacterial action was attributed to both bactericidal and bacteriostatic effects. Gene expression studies showed decreased expression of biofilm-related genes, indicating a specific anti-biofilm effect. Flow cytometric analyses revealed that AA leads to membrane hyperpolarization, altered membrane transport, increased membrane permeability, and membrane perforation. AA also acts as an antioxidant, and its anti-bacterial activity was counteracted by α-tocopherol, suggesting that oxidation of AA in bacteria produces cytotoxic radicals. Cytotoxicity studies showed that AA was non-toxic to normal Vero epithelial cells and did not cause hemolysis. The findings suggest that AA is a potentially safe drug for reducing the oral burden of *S. mutans*.The study investigates the anti-bacterial and anti-biofilm activities of arachidonic acid (AA) against *Streptococcus mutans*, a cariogenic bacterium. AA, a polyunsaturated fatty acid, was found to have a minimum inhibitory concentration (MIC) of 25 μg/ml in the presence of 5% CO2, reducing to 6.25–12.5 μg/ml without CO2. The anti-bacterial action was attributed to both bactericidal and bacteriostatic effects. Gene expression studies showed decreased expression of biofilm-related genes, indicating a specific anti-biofilm effect. Flow cytometric analyses revealed that AA leads to membrane hyperpolarization, altered membrane transport, increased membrane permeability, and membrane perforation. AA also acts as an antioxidant, and its anti-bacterial activity was counteracted by α-tocopherol, suggesting that oxidation of AA in bacteria produces cytotoxic radicals. Cytotoxicity studies showed that AA was non-toxic to normal Vero epithelial cells and did not cause hemolysis. The findings suggest that AA is a potentially safe drug for reducing the oral burden of *S. mutans*.