New rifamycin congeners (1-33) with incorporated amine and hydrazone substituents leading to lipophilic and/or basic nature and altered rigidity of modified C(3) arm were synthesized and structurally characterized in detail. NMR spectroscopic studies at different temperatures indicate two types of structures of rifamycin congeners that are realized in solution: zwitterionic and non-ionic forms in dependence of the basicity of modified C(3) arm. The presence of rifamycin congeners in these two possible forms has a significant impact on the physico-chemical parameters such as lipophilicity (clogP) and water solubility and different binding mode of the C(3) arm of antibiotic at RNAP binding pocket (molecular target) leading to different antibacterial potency. The highest antibacterial potency against S. aureus (including MRSA and MLSB strains) and S. epidermidis strains, even higher than reference rifampicin (Rif) and rifaximin (Rifx) antibiotics, was found for rifamycin congeners bearing at the C(3) arm relatively rigid and basic substituents (bipiperidine and guanidine groups). These modifications provide favorable docking mode and excellent water solubility resulting in high potency (MICs 0.0078 μg/mL what gives ∼ 8.5 nM), irrespective whether rifamycin congener is a tertiary amine (15) or hydrazone (29). In turn, for a higher antibacterial potency of rifamycin congeners against E. faecalis strain (MICs 0.5 μg/mL that is 0.6 μM) as compared to Rif and Rifx, the most crucial factors are: bulkiness and the lipophilic character of the end of the C(3) rebuilt arm.