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Objective: To synthesise the gold nanoparticles (AuNPs) using Acacia catechu through biogenic synthesis and evaluate their antimicrobial efficacy against S. mutans and E. coli in vitro. Methods: Green synthesised AuNPs were characterised using the ultraviolet–visible (UV-Vis) spectroscopy, and the size and shape of the synthesised nanoparticles were evaluated using the transmission electron microscopy (TEM). The antimicrobial efficacy of AuNPs (30/60/100 μl) against S. mutans/E. coli was evaluated on the Mueller–Hinton agar by measuring the zone of inhibition (ZOI) with ampicillin (15 μl) as a positive control. Results: The synthesised AuNPs were confirmed using the UV-Vis spectroscopy with peaks at 540 nm, and the size of the particle estimated using the TEM was between 5 and 15 nm. The antimicrobial efficacy of AuNPs was comparable to that of ampicillin against S. mutans/E. coli, but the difference was not significant. The antimicrobial effects increased in a dose-dependent fashion but were comparable across all concentrations and ampicillin. Conclusion: Green synthesised AuNPs exhibited significant antibacterial activity against S. mutans and E. coli at par with commercial ampicillin and demonstrated the potential towards anticariogenic agent for future use in dentistry.