Here, we report on a potent class of substituted ureidothiophenes targeting energy-coupling factor (ECF) transporters, an unexplored target that is not addressed by any antibiotic in the market. Since the ECF module is crucial for the vitamin transport mechanism, the prevention of substrate uptake should ultimately lead to cell death. By utilizing a combination of virtual and functional whole-cell screening of our in-house library, the membrane-bound protein mediated uptake of folate could be effectively inhibited. Structure-based optimization of our hit yielded low-micromolar inhibitors, whereby the most active compounds showed in addition potent antimicrobial activities against a panel of clinically relevant Gram-positive pathogens without significant cytotoxic effects.