In recent years, antimicrobial agents have been crucial in improving public health worldwide. However, due to improper usage, bacteria have developed resistance to these agents. This has led researchers to explore the use of heterocyclic compounds as alternative antimicrobial agents to combat resistance. Considering the success of heterocyclic derivatives in developing effective antimicrobial drugs, we conducted a 2D-QSAR (QSAR models using 2D-descriptors) study on novel pyrimidine derivatives and performed in silico simulations to evaluate their potential antibacterial activity against quinolone-resistant Escherichia coli. We used QSARINS V.2.2 (Insubria) software for the 2D-QSAR analysis and performed in silico absorption, distribution, metabolism, and excretion (ADME) and docking studies on 16 newly designed compounds. The best QSAR model had a high correlation coefficient ([Formula: see text], [Formula: see text], [Formula: see text]) and showed no outliers. Based on these favorable results, we designed 16 new compounds and predicted their antibacterial activity using the best equation. Amongst the top six designed compounds, which had the best docking scores were subjected to practical synthesis. Compound 3cd was found to be good candidature from in-vitro anti-E. Coli activity. This compound may be targeting the DNA gyrases and thus, have inhibitory activity against E. Coli. This observation was also supported by 100[Formula: see text]ns molecular dynamics and normal mode analysis results. The most promising compounds identified through these computational studies may be synthesized and tested as potential new drug candidates for treating bacterial infections.