Molecules that target the deoxyribonucleic acid (DNA) minor groove are relatively sequence specific and they can be excellent carrier structures for cytotoxic chemotherapeutic compounds which can help to minimize side effects. Two novel isomeric derivatives of diaminobenzene Schiff base [N,N'-bis (2-hydroxy-3-methoxybenzylidene)-1,2-diaminobenzene (2MJ) and N,N'-bis(2-hydroxy-3-methoxybenzylidene)-1,3-diaminobenzene (2MH)] were analyzed for their DNA minor groove binding (MGB) ability using viscometry, UV and fluorescence spectroscopy, computational modeling and clonogenic assay. The result shows that 2MJ and 2MH are strong DNA MGBs with the latter being more potent. 2MH can form interstrand hydrogen bond linkages at its oxygens with N3 of adenines. Changing the 2-hydroxy-3-methoxybenzylidene binding position to the 1,3 location on the diaminobenzene structure (2MJ) completely removed any viable hydrogen bond formation with the DNA and caused significant decrease in binding strength and minor groove binding potency. Neither compound showed any significant cytotoxicity towards human breast, colon or liver cancer cell lines.