Graphical abstract A series of rhodanine derivatives RB1–RB23 were synthesized through a two-round screening with their Mycobacterial tuberculosis (Mtb) InhA inhibitory activity and Mtb growth blocking capability evaluated. The improved growth-blocking effect on Mtb and low toxicity were attractive for further development. The docking simulation the key interacted residues as Ser20, Phe149, Lys165 and Thr196 in the binding pattern. Highlights • 23 Rhodanine derivatives were synthesized through a two-round screening. • Their anti-bacterial activity was evaluated for the first time. • The top hit showed improved growth-blocking effect on Mtb and low toxicity. • Molecular docking indicated possible binding pattern and key interacted residues. Abstract A series of rhodanine derivatives RB1–RB23 were synthesized through a two-round screening. Their Mycobacterial tuberculosis (Mtb) InhA inhibitory activity and Mtb growth blocking capability were evaluated. The most potent hit compound RB23 indicated comparable InhA inhibiton (IC 50 = 2.55 μM) with the positive control Triclosan (IC 50 = 6.14 μM) and Isoniazid (IC 50 = 8.29 μM). Its improved growth-blocking effect on Mtb and low toxicity were attractive for further development. The docking simulation revealed the possible binding pattern of this series and picked the key interacted residues as Ser20, Phe149, Lys165 and Thr196. The 3D-QSAR model visualized the SAR discussion and hinted new information. Modifying the surroundings near rhodanine moiety might be promising attempts in later investigations. [ABSTRACT FROM AUTHOR]