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Chitosan-grafted thymol (CST) coated on gold nanoparticles has been synthesized and characterized for the design of antimicrobial materials. CST was synthesized via adapting the Mannich reaction, and it acted as the capping agent for the synthesis of gold nanoparticles (AuNPs). The grafting of thymol onto the side chain of chitosan has provided a degree of substitution value (%DS NMR ) of 10.0%, calculated by nuclear magnetic resonance spectroscopy. UV–visible spectrometry and elemental analysis were used to confirm the successful synthesis of CST through adapting the Mannich reaction. The appropriate concentration of CST for AuNP synthesis was found to be 0.020%w/v. A red-wine colloidal AuNP solution of 2.41–3.30 nM particle size exhibits a strong surface plasmon resonance at 502 nm, which shows negative charges at pH = 9 of −36.37 mV. This result evidenced that the AuNPs showed electrostatic repulsion and CST played a role as a capping agent to provide a good dispersion and stability state. CST coated on the AuNP surface was successfully utilized for the control of cariogenic bacteria in the oral cavity. The results obtained from this study show that the tuning of the capping agent used in the synthesis step strongly influences the latter antimicrobial activity of the nanoparticles against Streptococcus mutans ATCC 25175 and Streptococcus sobrinus ATCC 33402 activity, with an inhibition zone of 15.90 and 14.25 mm, respectively. The average minimum inhibitory concentration values against S. mutans ATCC 25175 and S. sobrinus ATCC 33402 were found to be 25 and 100 mg/L, respectively, whereas the minimum bactericidal concentration values were 100 and 200 mg/L, respectively.