Seven new benzyl (3–9) and four new phenethyl (10–13) amino analogues of ansa-macrolide rifampicin (1) were synthesized using the optimised method of reductive amination. Structures of 3–13 in solution were determined by 1D and 2D NMR and FT-IR methods whereas the energetically most favoured conformation of amino analogues was calculated with the use of the PM5 method. Spectroscopic and semi-empirical studies revealed the presence of zwitterionic forms of all 3–13 analogues in solutions containing water traces. ¹H–¹⁵N HSQC and ¹H–¹⁵N HMBC in combination with ¹H–¹H COSY and ¹H–¹³C HMBC two dimensional spectroscopic methods unambiguously evidenced that the presence of the zwitterionic form of ansa-macrolides was a consequence of proton transfer from the O(8)–H phenolic group to the secondary amine moiety within 3–13 structures. ¹H–¹H NOESY studies indicated two different orientations of the substituent introduced at the C(3) position for benzyl and phenethyl amino analogues of rifampicin and their similar conformation within the ansa-bridges in solution. FT-IR studies of the deprotonation of molecule 1 and comparison of these data with those for 3–13 indicated C(8)O double bond character after formation of zwitterions in solution. Results of an antibacterial test against Gram-(−) and Gram-(+) strains were compared with detailed structural information on new analogues of 3–13 to indicate some structure–activity relationships. Molecular recognition studies of 1 and 12 inhibitors at the binding site of bacterial RNA polymerase (RNAP) as zwitterions revealed key intermolecular interactions and led to the proposition of a new model of RNAP inhibition, which explains significant differences in antibacterial properties of rifampicin and its analogues.