Silver nanoparticles (AgNPs) gain great interest among noble metal nanoparticles due to their broad applications in medicine, dentistry, drug delivery, tissue and tumour imaging, biolabeling, and biosensing. The antibacterial, antifungal, antiviral, and antiparasitic activity of AgNPs is well documented in the literature. This study aimed to determine the antimicrobial and cytotoxic activity of Neem and Kirata herbal formulation-mediated silver nanoparticles against oral biofilm. The green synthesis of Neem and Kirata herbal formulation-mediated silver nanoparticles was done. The antimicrobial action against the strains of Candida albicans, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans were assessed. The results showed that the newly formulated nanoparticle had effective anti-microbial properties and decreased cytotoxic properties which make it advantageous for clinical applications and treatment modalities. It showed great potential for the nanoparticle in decreasing the bacterial effects and cytotoxic nature thereby providing future scope for clinical application in preventing oral biofilm formation and its deleterious effects.