Bacteria are the most abundant life form on Earth. A functional understanding of the genetic factors underpinning bacterial success has important medical, economic, and ecological implications. As increasingly cost-efficient sequencing technologies have led to the sequencing of exponential bacterial genomes, it is critical to develop new functional methodologies to assign functions to these newly sequenced genes at a comparable pace. Advancements in genomewide mutant library construction techniques have driven the development of such functional techniques. Here, I introduce this thesis with a review of how mutant library construction reflects functional assay readouts. In Chapter 2, important considerations and corrections are presented for one of these new high-throughput functional assays: pooled CRISPRi chemical genomics. In Chapter 3, I present a high-throughput, whole-genome chemical genomics screen in Bacillus subtilis, the model gram-positive organism. This screen highlights the power of these new technologies to uncover novel biology and will act as a blueprint for similar screens in other bacterial species. Finally, in Chapter 4, I summarize the contributions of this thesis to the field of bacterial functional genomics and explore future applications of this exciting approach.