Topoisomerases (topos) are enzymes responsible for managing DNA topology and linking, making them critical cellular machinery for the management of DNA. Due to their key roles in cellular maintenance and replication, topo inhibitors are used clinically as antimicrobials and anticancer agents. Fluoroquinolones are clinically successful bacterial type II topo inhibitors or poisons used to treat bacterial infections and several clinically successful cancer chemotherapy agents are inhibitors or poisons of human topos. Quinazoline-2,4-diones (diones) are structural mimics of fluoroquinolones that may mimic the topo inhibiting activity of either antibacterial or anticancer fluroquinolones. These diones show promise both as antimicrobials for antibiotic-resistant bacteria or as anticancer chemotherapy agents, but for both applications there is an apparent problem with cellular accumulation of diones. This work describes the exploration of two strategies for increasing the cellular accumulation of antibiotic or anticancer diones. In the clinic, fluoroquinolones face both target-mediated and plasmid-mediated resistance (efflux resistance). Quinazoline 2,4-dione dimers were proposed as a solution to both mechanisms of resistance. Past work in the Kerns lab has shown that C7-linked fluoroquinolone dimers overcome efflux-based fluoroquinolone resistance in Gram-positive bacteria with upregulated efflux pumps while maintaining or having improved antibacterial activity with different Gram-positive organisms. It was hypothesized that dione dimers would also maintain antibacterial activity and evade efflux. In pursuit of these goals, three C7-piperazenyl-linked antimicrobial dione dimers and one C7-linked dione/fluoroquinolone asymmetric dimer were synthesized, and all but the asymmetric dimer were poor bacterial topo inhibitors. N1-biphenyl fluoroquinolones are human topo inhibitors with unique properties that set them apart from conventional fluoroquinolones and anticancer fluoroquinolones. Unlike conventional fluoroquinolones, N1-biphenyl fluoroquinolones target type I topos over type II topos and are catalytic inhibitors rather than topo poisons. N1-biphenyl diones are the same and have equal human topo I inhibition to N1-biphenyl fluoroquinolones. Despite the similar topo I inhibition of a representative fluoroquinolone and a representative dione, the results of NCI 60 cancer cell lines screening were significantly different. In multiple cell lines N1-biphenyl fluoroquinolones had greater growth inhibition than N1-biphenyl diones. This suggests that there is a difference in cellular uptake between the fluoroquinolone and dione. It was hypothesized that this difference was related to the charge and physical properties of these diones and quinolones. A panel of N1-biphenyl diones with altered charged groups at C7 and N3 were synthesized to evaluate whether changes in the charged groups at these locations could alter the ability of diones to enter cells, evade efflux, and inhibit topo I. The diones were predicted to have lower partition coefficients than their cognate quinolones. Topo I DNA relaxation, cell viability, and DNA binding were evaluated for these compounds. It was determined that DNA binding, enzyme inhibition, and cell viability inhibition track well together, which suggests that, at least in the cell types studied, cell penetration does not appear to be a major issue. The DNA binding assays also confirm that DNA binding is an important factor in topo I inhibition for these compounds.