Dental caries is a biofilm-dependent disease, and Streptococcus mutans is the primary etiological agent involved in the initiation of the disease. The extensive use of a limited range of antimicrobial drugs in dentistry has led to the development of drug-resistant bacteria. There is an increasing need to find new alternatives against drug-resistant bacteria. Globally, there is a continuous effort towards identifying natural anti-caries agents for the prevention and better management of caries. The objective of the present study was to evaluate the antibiofilm potential of Azadirachta indica leaf methanolic (ALM) extract against S. mutans biofilm. The study employed a standard reference strain of S. mutans MTCC 497, for in vitro standardisation of biofilm by microtiter plate assay. The antibiofilm activity of the ALM extract was evaluated against the S. mutans strain, and the same was confirmed by light and scanning electron microscopy (SEM). The in vitro biofilm standardisation results demonstrated that 50 µl/ml of S. mutans inoculum concentration exhibited a much superior biofilm formation than the other concentrations employed. Light microscopy and SEM images revealed that ALM extract at 100 mg/ml concentration significantly inhibited the S. mutans biofilm. To conclude, the study reports that the A. indica leaf extract is a potential source to inhibit the S. mutans biofilm. Further studies are warranted to identify the phytochemicals responsible for the antibiofilm activity of ALM extract against S. mutans biofilm that aid in the design of natural anti-caries products.