Over the last decade, our understanding of isoprenoid biosynthesis has progressed to the stage where specific strategies for the bioengineering of essential oil production can be considered. This review provides a current overview of the enzymology and regulation of essential oil isoprenoid biosynthesis. The reaction mechanisms of the synthases which produce many of the basic isoprenoid skeletons are described in detail. Coverage is also provided of the regulation of isoprenoid biosynthesis, including the roles played by tissue and subcellular compartmentation, and by partitioning of intermediates between different branches of isoprenoid metabolism. This provides necessary context for rationally targeting specific enzymes of metabolic pathways for bioengineering essential oil production. Wherever possible, emphasis is placed on research specific to essential oil isoprenoid biosynthesis, although relevant work related to other isoprenoids is also considered when it can provide useful insights. Finally, building upon this understanding of essential oil isoprenoid biosynthesis, several approaches to the bioengineering of isoprenoid metabolism are considered.