Aim: Antibiotic resistance has become a major health problem. The σ70:core interface of bacterial RNA polymerase is a promising drug target. Recently, the coiled-coil and lid-rudder-system of the β’ subunit has been identified as an inhibition hot spot. Materials & methods & Results: By using surface plasmon resonance-based assays, inhibitors of the protein–protein interaction were identified and competition with σ70 was shown. Effective inhibition was verified in an in vitro transcription and a σ70:core assembly assay. For one hit series, we found a correlation between activity and affinity. Mutant interaction studies suggest the inhibitors’ binding site. Conclusion: Surface plasmon resonance is a valuable technology in drug design, that has been used in this study to identify and evaluate σ70:core RNA polymerase inhibitors.