Bacterial infections present a serious challenge to healthcare practitioners due to the emergence of resistance to numerous conventional antibacterial drugs. Therefore, new bacterial targets and new antimicrobials are unmet medical needs. Rhodanine derivatives are known to possess potent antimicrobial activities. In this study, we determined the activity spectrum of a series of new rhodanine derivatives against representative Gram-positive and Gram-negative bacterial strains. Compounds 3a and 5a had the highest activity with minimum inhibitory concentrations in the range of 1.12-2.5 μg/mL. Transmission electron microscope results confirmed that activities against bacteria occurred via rupturing of the cell wall. Molecular modeling results suggested that rhodanine derivatives have the potential to irreversibly bind to the penicillin-binding protein (PBP) Ser62 residue in the active site. Thus, our results suggested that these rhodanine derivatives could be potential antibacterial drug candidates with strong activity against Gram-negative bacteria.