Antibacterial minor groove binders related to the natural product, distamycin, are development candidates for novel antibiotics. Alkenes have been found to be effective substitutes for the isosteric amide links in some positions and alkyl groups larger than methyl have been found to increase binding to DNA in both selectivity and affinity. However the impact of other isosteres such as diazenes and the position of an alkyl group with respect to DNA binding and antibacterial activity are not known. The effects of some systematic variations in the structure of polyamide minor groove binders are investigated. Isosteres of the amide link (alkenes and diazenes) are compared: it is shown that all three are competent for binding to DNA but that alkene links give the tightest binding and highest antibacterial activity; no significant antibacterial activity was found for compounds with a diazene link. Within a series of alkene linked compounds, the effect of branched N-alkyl substituents on binding to DNA and antibacterial activity is investigated: it was found that C3 and C4 branched chains are acceptable at the central pyrrole residue but that at the pyrrole ring adjacent to the basic tail group, a C4 branched chain was too large both for DNA binding and for antibacterial activity. The active branched alkyl chain compounds were found to be especially active against Mycobacterium aurum, a bacterium related to the causative agent of tuberculosis.