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Pathogenic <i>Escherichia coli</i> cannot be killed by most antibiotics (including colistin, a last-resort drug) due to the rapid development of antibiotic resistance. A highly conserved prokaryotic mitotic protein, filamenting temperature-sensitive protein Z (FtsZ) with GTPase activity, plays a key role in cell division and has become a promising target for screening novel antibacterial agents. In this study, the amplified <i>ftsZ</i> gene was inserted into cloning/expression vectors and recombinantly produced in <i>E. coli</i>; the recombinant FtsZ protein was purified by the Ni²⁺-NTA affinity column and then was used to screen for natural antibacterial agents. The results showed that the <i>ftsZ</i> gene with a size of 1170 bp was successfully amplified from <i>E. coli</i> and inserted into the pET-28a expression vector. After induction with 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), FtsZ was expressed in <i>E. coli</i> BL21 as inclusion bodies. After purification, the recombinant FtsZ protein showed GTPase activity. The highest GTPase activity (0.998 nmol/mL/min) of FtsZ was observed at a GTP concentration of 1.25 mM. Several alkaloids were screened by a constructed model of FtsZ inhibitors. Sanguinarine chloride exhibited higher antibacterial activity against <i>E. coli</i> and <i>Salmonella enteritidis</i> (with minimum inhibitory concentrations (MICs) of 0.04–0.16 mg/mL and minimum bactericidal concentrations (MBCs) of 0.16–0.32 mg/mL) than tetrandrine (0.16–0.32 mg/mL) and berberine hydrochloride (0.32–0.64 mg/mL). Berberine hydrochloride prevented FtsZ polymerization in a concentration-dependent manner and bound to FtsZ protein by hydrogen bonding interaction. This study suggested that the FtsZ-based <i>E. coli</i> screening model could be exploited for the development of novel antibacterial agents for clinical applications.