Adeno-associated virus (AAV) is a promising vector for human gene therapy. Although more effective than non-viral vectors, AAV still requires improvement in efficacy in order to become a successful gene therapy vector. With this in mind, we have sought to identify and examine identified enhancers of adeno-associated virus type 2 (AAV2) transduction. Using a high throughput screening system with recombinant AAV2 carrying the luciferase reporter gene (AAV2-Luc), we found siRNA sequences and chemical compounds which increase AAV2 reporter gene expression. We specifically identified a hexamer seed region 5’-UGUUUC-3’ which facilitated AAV2 transduction. Chemical compound enhancers included ellagic acid, 1,10-phenanthroline, EGFR tyrosine kinase inhibitors, nucleoside analogs, and DNA alkylating agents. Although several of these compounds, such as EGFR tyrosine kinase inhibitors and DNA alkylating agents, were known enhancers of AAV transduction, compounds such as ellagic acid and 1,10-phenanthroline were newly identified as facilitating AAV2 transduction. After identifying these enhancers, we have further sought to understand a mechanistic basis for them through studies which individually quantified enhancement at stages including the virus-receptor interaction, the viral DNA introduction into the cell, reporter gene RNA transcription, and the production of protein from the transgene. The identification of siRNAs and chemical compounds which enhance transduction can lead to a better understanding of AAV2 biology and may provide a foundation for the engineering of novel AAV formulations, delivery systems, or vectors.