Arylamine N-acetyltransferase 1 (NAT1) expression has been shown to attenuate mitochondrial function, suggesting it is a promising drug target in diseases of mitochondrial dysfunction. Here, several second-generation naphthoquinones have been investigated as small molecule inhibitors of NAT1. The results show that the compounds inhibit both in vitro and in whole cells. A lead compound (Cmp350) was further investigated for its ability to alter mitochondrial metabolism in MDA-MB-231 cells. At concentrations that inhibited NAT1 by over 85%, no overt toxicity was observed. Moreover, the inhibitor decreased basal respiration and reserve respiratory capacity without affecting ATP production. Cells treated with Cmp350 were almost exclusively dependent on glucose as a fuel source. We postulate that Cmp350 is an excellent lead compound for the development of NAT1-targeted inhibitors as both experimental tools and therapeutics in the treatment of hypermetabolic diseases such as amyotrophic lateral sclerosis, cancer cachexia, and sepsis.