Formation of a bacterial RNA polymerase (RNAP) holoenzyme by a catalytic core RNAP and a sigma (σ) initiation factor is essential for bacterial viability. As the primary binding site for the housekeeping σ factors, the RNAP clamp helix domain represents an attractive target for novel antimicrobial agent discovery. Previously, we designed a pharmacophore model based on the essential amino acids of the clamp helix, such as R278, R281, and I291 (<i>Escherichia coli</i> numbering), and identified hit compounds with antimicrobial activity that interfered with the core-σ interactions. In this work, we rationally designed and synthesized a class of triaryl derivatives of one hit compound and succeeded in drastically improving the antimicrobial activity against <i>Streptococcus pneumoniae</i>, with the minimum inhibitory concentration reduced from 256 to 1 μg/mL. Additional characterization of antimicrobial activity, inhibition of transcription, in vitro pharmacological properties, and cytotoxicity of the optimized compounds demonstrated their potential for further development.