Introduction Bacteria associated with dental caries have a high ability to produce organic acids from dietary carbohydrates during growth and metabolism under acidic conditions. In contrast, many symbiotic bacteria produce ammonia through the arginine deiminase (ADS) system, which modulates the pH of the oral cavity. l-Arginine metabolism by ADS is a significant inhibitor in the progression of tooth decay. This study aimed to investigate the effect of l-arginine on growth, biofilm formation, and antibiotic susceptibility in Streptococcus mutans. Methods In this study, the effect of l-arginine in different concentrations on the growth rate, antibiotic susceptibility, and inhibition of biofilm formation in S. mutans was investigated. Results The bacterial exponential growth rate was enhanced by 100 μM l-arginine (P > 0.05). The growth inhibition zone diameter of CAZ, CTR, AMP, and AMC-Clav antibiotics was reduced after 24 h of exposure in the presence of various concentrations of l-arginine specifically at 100 μM. l-Arginine also enhanced biofilm development at 5 and 10 μM concentrations, but reduced it at 50 and 100 μM concentrations. Conclusion According to the results of the present study, optimization of l-arginine concentration and its use as an adjunctive therapy or in combination with mouthwash or varnish is recommended to prevent oral caries.