Bridging high-throughput screening (HTS) with RNA interference (RNAi) has allowed for rapid discovery of the molecular basis of many diseases, and identification of potential pathways for developing safe and effective treatments. These features have identified new host gene targets for existing drugs paving the pathway for therapeutic drug repositioning. Using RNAi to discover and help validate new drug targets has also provided a means to filter and prioritize promising therapeutics. This review summarizes these approaches across a spectrum of methods and targets in the host response to pathogens. Particular attention is given to the utility of drug repurposing utilizing the promiscuous nature of some drugs that affect multiple molecules or pathways, and how these biological pathways can be targeted to regulate disease outcome.