Amid grave concerns over global climate change and with increasingly strained access to fossil fuels, the synthetic biology community has stepped up to the challenge of developing microbial platforms for the production of advanced biofuels. The adoption of gasoline, diesel, and jet fuel alternatives derived from microbial sources has the potential to significantly limit net greenhouse gas emissions. In this effort, great strides have been made in recent years toward the engineering of microorganisms to produce transportation fuels derived from alcohol, fatty acid, and isoprenoid biosynthesis. We provide an overview of the biosynthetic pathways devised in the strain development of biofuel-producing microorganisms. We also highlight many of the commonly used and newly devised engineering strategies that have been employed to identify and overcome pathway bottlenecks and problems of toxicity to maximize production titers.