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Biofilm is formed by one or different types of microbes that can grow on different surfaces. Bacteria, fungi, and many protists can form biofilms. Biofilm formation requires cell to cell signaling mechanism which is defined as quorum sensing. In this work, we have studied about the antibiofilm activity of methanolic extract of nutmeg against Gram-positive Bacillus subtilis bacteria. Our test extract inhibited biofilm production as well as eradication of pre-formed biofilm in B. subtilis. Extracellular polymeric substance (EPS) is the biopolymer of microbial origin in which biofilm producing microbes are embedded. When test extract was treated with organisms producing biofilms it can also reduce the production of EPS of biofilm. All the quantitative analysis of this work concludes that methanolic extract of Myristica fragrans seed extract inhibited biofilm formation by 47%, eradicated the pre-formed biofilm by 28.5%, and inhibited EPS formation by 60%. All these quantitative tests were confirmed by microscopic analysis. All Our study concludes that M. fragrans seed extract can inhibit biofilm formation and can destruct preformed biofilm in Gram-positive bacteria.