Polymethyl-methacrylate (PMMA) is commonly used primarily for dentures and orthodontic appliances. The activities of cariogenic bacteria in biofilm on these surfaces may contribute to dental caries, gingival inflammation and periodontal disease. Nanoparticles of silver (NanoAg) have been shown to control the growth of bacteria, but anti-cariogenic application of NanoAg in situ in PMMA (NanoAg-IS-PMMA) in orthodontics has not been evaluated. This in vitro study evaluates the antimicrobial activity of NanoAg-IS-PMMA against four cariogenic bacteria. Chemical-cure orthodontic resin was used to synthesize NanoAg-IS-PMMA using silver benzoate. Antibacterial effectiveness of NanoAg-IS-PMMA was assessed against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus as well as Lactobacillus casei and mixed cultures of the four species by adherence inhibition as well as planktonic and biofilm bacterial cells growth inhibition on NanoAg-IS-PMMA. Exposure to NanoAg-IS-PMMA reduced bacterial adherence by 29.6-90.8% depending on the microorganism type. Planktonic cultures of cariogenic bacteria exposed to NanoAg-IS-PMMA showed 99.99% decrease in timedependent manner over a 28 day period. Biofilm inhibition analysis demonstrated that NanoAg-IS-PMMA inhibited the biofilm of all test bacteria and mixed organism’s cultures by 78.1-99.7%, compared to PMMA. NanoAg-ISPMMA maintained at least some inhibitory activity even after the third generation of biofilm growth. In conclusion, the data presented here are novel in that they prove that NanoAg-IS-PMMA effectively inhibited adherence of cariogenic bacteria to NanoAg-IS-PMMA surfaces as well as strong antimicrobial activity in the planktonic phase and subsequent biofilm formation. This showed NanoAg-IS-PMMA has the potential to minimize cariogenic microorganism’s colonization on orthodontic appliances.