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Biofilms currently represent the most prevalent bacterial lifestyle, enabling them to resist environmental stress and antibacterial drugs. Natural antibacterial agents could be a safe solution for controlling bacterial biofilms in food industries without affecting human health and environmental safety. A methanolic extract of <i>Azadirachta indica</i> (neem) leaves was prepared and analyzed using gas chromatography–mass spectrometry for the identification of its phytochemical constituents. Four food-borne bacterial pathogens (<i>Bacillus cereus</i>, <i>Novosphingobium aromaticivorans</i>, <i>Klebsiella pneumoniae</i>, and <i>Serratia marcescens</i>) were tested for biofilm formation qualitatively and quantitatively. The antibacterial and antibiofilm properties of the extract were estimated using liquid cultures and a microtiter plate assay. The biofilm inhibition mechanisms were investigated using a light microscope and molecular docking technique. The methanolic extract contained 45 identified compounds, including fatty acids, ester, phenols, flavonoids, terpenes, steroids, and antioxidants with antimicrobial, anticancer, and anti-inflammatory properties. Substantial antibacterial activity in relation to the extract was recorded, especially at 100 μg/mL against <i>K. pneumoniae</i> and <i>S. marcescens</i>. The extract inhibited biofilm formation at 100 μg/mL by 83.83% (<i>S. marcescens</i>), 73.12% (<i>K. pneumoniae</i>), and 54.4% (<i>N. aromaticivorans</i>). The results indicate efficient biofilm formation by the Gram-negative bacteria <i>S. marcescens</i>, <i>K. pneumoniae</i>, and <i>N. aromaticivorans</i>, giving 0.74, 0.292, and 0.219 OD at 595 nm, respectively, while <i>B. cereus</i> was found to have a low biofilm formation potential, i.e., 0.14 OD at 595 nm. The light microscope technique shows the antibiofilm activities with the biofilm almost disappearing at 75 μg/mL and 100 μg/mL concentrations. This antibiofilm property was attributed to DNA gyrase inhibition as illustrated by the molecular docking approach.