AbstractThe rise of multidrug‐resistant organisms (MDROs) represents a significant challenge to healthcare, underscoring the need for novel antimicrobial agents. Quinazolinone compounds, noted for their diverse biological activities, particularly at the 2nd and 3rd positions, in conjunction with sulphanilamide and isatin derivatives, present a promising avenue for antibacterial development. This study focuses on the synthesis of novel 2,3‐disubstituted quinazolin‐4(3H)‐one derivatives from Schiff base intermediates GA3A and GA3B. The synthesis involved the reaction of 2‐(substituted phenyl)‐4H‐benzo[d][1,3]‐oxazin‐4‐one with sulphanilamide, and the benzoxazinone intermediates were prepared by reacting anthranilic acid with benzoyl chloride. The antibacterial activities of the Schiff base intermediates and the final Mannich base compounds were evaluated against Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa at concentrations of 50 µg/mL and 100 µg/mL using the agar well diffusion method, with Norfloxacin (50 µg/mL) as the reference standard. While all tested compounds exhibited lower antibacterial activity compared to the standard, GA4A1 showed enhanced efficacy against E. coli, achieving the highest docking score of 78.0352 against the E. coli protein (PDB ID: 1KZN). Molecular dynamics simulations revealed that the GA4A1‐E. coli complex stabilized after 40,000 ps, with root mean square deviation (RMSD) values ranging from 2.5 Å to 5 Å and low root mean square fluctuation (RMSF) values between 0.05 Å and 0.2 Å, indicating the stability of the complex. These findings underscore GA4A1’s potential as a potent antimicrobial agent against E. coli.