Diamidines are a class of compounds that target the minor groove of DNA and have antiparasitic and antimicrobial properties. Their mechanism of action has not been fully elucidated, but may include changes in DNA topology. In this study we have investigated such changes using methods of gel electrophoresis including ligation ladders and cyclization assays. We found that topology changes were sequence dependent. Compounds typically caused non-anomalously migrating ATATA sequences to migrate as if they were bent, while A5 sequences that normally migrated anomalously became less so in the presence of certain diamidines. Select compounds induced changes in cyclization efficiency that were also sequence dependent; DB75 significantly abolished cyclization in A5 containing sequences but enhanced it in sequences containing ATATA sites.