Background: Dental caries formation is highly prevented by applying pit and fissure sealant but was associated with microleakage and secondary caries formation. Aim and Objectives: To synthesize novel pit and fissure sealant with green synthesized silver nanoparticles and evaluation of antibacterial activity against Streptococcus mutans, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans. Materials and Methods: Green synthesis of silver nanoparticles was done using beetroot, characterized using FTIR (Fourier transform infrared), EDAX (Energy Dispersive X-ray Analysis), and Scanned Electron Microscope Analysis. A novel pit and fissure sealant was made by incorporating synthesized Ag nanoparticles and hydrophilic nature from Ultra seal XT Hydro. Using amoxicillin as a control, an agar well diffusion method was used to conduct an antimicrobial test for a new sealant against Staphylococcus aureus, Streptococcus mutants, Enterococcus faecalis, and Candida albicans in Muller Hinton Agar \((n=3)\) for each organism. Result: Zone of inhibition was measured, showing statistically significant antimicrobial activity against Streptococcus mutans with the ZOI value \(21mm \pm 2mm\) at \(100\mu l\) concentration of Ag nanoparticles in sealant material. Conclusion: Novel Hydrophilic pit and fissure sealant with Ag nanoparticles are potentially antibacterial against the cariogenic microorganism. Thus, it can be utilized for the better efficiency of topical pit and fissure sealant preventive therapy.