The assembly and disassembly of FtsZ play an essential role in bacterial cell division. Using single‐cell imaging, we report that short exposure to BT‐benzo‐29 inhibits Z‐ring formation in live Bacillus subtilis cells. Fluorescence recovery after photobleaching of the Z‐ring in live bacteria demonstrated that BT‐benzo‐29 strongly suppressed the assembly dynamics of FtsZ in the Z‐ring. Furthermore, B. subtilis cells expressing V275A‐FtsZ resisted the antibacterial activity of BT‐benzo‐29 providing evidence that BT‐benzo‐29 inhibits bacterial proliferation by targeting FtsZ. In addition, a brief (8 min) exposure of BT‐benzo‐29 destroyed the Z‐ring without perturbing the localization of a late cell division protein, DivIVA, the nucleoid segregation, and membrane permeability. BT‐benzo‐29, when used in combination with vancomycin and polymyxin B (PMB), produced a much stronger inhibitory effect on Bacillus subtilis and Escherichia coli cells, respectively. The combination index of BT‐benzo‐29 with vancomycin and PMB was determined to be <1, suggesting that BT‐benzo‐29 exhibits synergistic inhibitory effects on bacterial proliferation when used along with these antibiotics.