Fluorescent microgonotropens (FMGTs) are A/T selective, minor groove-binding bisbenzimidazole ligands. Basic side chains extending from these agents electrostatically contact the major groove side of the phosphodiester backbone of DNA, endowing them with high binding affinity. Here, we evaluate the potential of these agents as inhibitors of transcription factor (TF) binding to DNA and explore whether their ability to contact both grooves enhances their inhibitory activity. A series of FMGTs (L2-L5), with polyamine tails of varying lengths and degrees of branching, were compared to an analog lacking these basic side chains (L1), and the classical bisbenzimidazole Hoechst 33342 for effects on TF complex formation on the c-fos serum response element (SRE). Although L1 could not inhibit TF/SRE interactions, L2-L5 did so at submicromolar concentrations. Moreover, the FMGTs were up to 50 times more potent than Hoechst 33342 in inhibiting TF complex formation in electrophoretic mobility shift assays. The FMGTs also inhibited c-fos promoter-driven cell-free transcription and topoisomerase II activity in nuclei. These studies establish the potential of FMGTs as TF/DNA complex inhibitors in cell-free systems, provide insight into the relationship between their structure and biological activities, and demonstrate the benefits of functionalizing minor groove binding-agents with major groove-contacting groups.