Medium-chain fatty acids (MCFAs; C6–C12) are valuable molecules used for biofuel and oleochemical production; however, it is challenging to synthesize these fatty acids efficiently using microbial biocatalysts due to the cellular toxicity of MCFAs. In this study, both the endogenous fatty acid synthase (FAS) and an orthogonal bacterial type I FAS were engineered for MCFA production in the yeast Saccharomyces cerevisiae. To improve cellular tolerance to toxic MCFAs, we performed directed evolution of the membrane transporter Tpo1 and strain adaptive laboratory evolution, which elevated the MCFA production by 1.3 ± 0.3- and 1.7 ± 0.2-fold, respectively. We therefore further engineered the highly resistant strain to augment the metabolic flux towards MCFAs. This multidimensional engineering of the yeast at the single protein/enzyme level, the pathway level and the cellular level, combined with an optimized cultivation process, resulted in the production of >1 g l−1 extracellular MCFAs—a more than 250-fold improvement over the original strain.