Here, we report on the design, synthesis and biological evaluation of 4-thiazolidinone (rhodanine) de-rivatives targeting Mycobacterial tuberculosis (Mtb) trans-2-enoyl-acyl carrier protein reductase (InhA). Compounds having bulky aromatic substituents at position 5 and a tryptophan residue at position N-3 of the rhodanine ring were the most active against InhA with IC50 values ranging from 2.7 µM to 30 µM. The experimental data showed consistent correlations with computational studies. Their anti-microbial activity was assessed against Mycobacterium marinum (Mm) (a model for Mtb), Pseudomonas aeruginosa (Pa), Legionella pneumophila (Lp) and Enterococcus faecalis (Ef), by using anti-infective, anti-virulence and antibiotic assays. 19 out of 34 compounds reduced Mm virulence at 10 µM. 33 exhibited promising antibiotic activity against Mm with a MIC of 0.21 µM and showed up to 89% reduction of Lp growth in an anti-infective assay at 30 µM. 32 showed high antibiotic activity against Ef with a MIC of 0.57 µM.