OBJECTIVE:The study investigated the effect of incorporating l-arginine (Arg) in a glass ionomer cement (GIC) on its mechanical properties and antibacterial potential. METHODS:Pre-determined proportions (1%, 2%, and 4% by wt.) of Arg were incorporated in GIC powder; while GIC without Arg served as control. The flexural strength, nanohardness, surface roughness, elemental analysis using SEM-EDX (n = 6) and F/Arg/Ca/Al/Si release in deionized water for 21 days were assessed. The antibacterial potential was evaluated in a multi-species biofilm model with Streptococcus mutans, Streptococcus sanguinis, Streptococcus gordonii, and Lactobacillus acidophilus for 72 h. Real-time qPCR was used to analyse biofilm bacterial concentrations. Propidium monoazide modification of real-time qPCR was performed to quantify viable/dead bacteria. The pH, lactic acid, ADS activity, and H2O2 metabolism were measured. Confocal microscopy was used to investigate the biofilm bacterial live/dead cells, density, and thickness. RESULTS:There was no difference in flexural strength among the different groups (p > 0.05). No significant difference in nanohardness and surface roughness was observed between 4% Arg + GIC and control (p > 0.05). The 4% Arg + GIC showed significantly higher F/Arg/Al/Si release than the other groups (p < 0.05), reduced total bacterial concentration and growth inhibition of viable S. mutans and S. sanguinis (p < 0.05). Lactic acid formation for 4% Arg + GIC was significantly higher than 1% Arg + GIC (p < 0.05). The spent media pH of 4% Arg + GIC was higher than the other groups (p < 0.05), with proportionately lower ammonia and higher H2O2 released (p < 0.05). SIGNIFICANCE:Addition of 4% l-arginine in GIC enhanced its antibacterial activity via a biofilm modulatory effect for microbial homeostasis, with no detrimental effect on its mechanical properties.