Cinnamon bark (Cinnamomum zeylanicum) is used extensively as an antimicrobial material and currently is being increasingly used in Europe by people with type-II diabetes to control their glucose levels. In this paper we describe the action of cinnamon oil, its major component, trans-cinnamaldehyde, and an analogue, 4-hydroxy-3-methoxy-trans-cinnamaldehyde against bacterial acetyl-CoA carboxylase in an attempt to elucidate the mechanism of action of this well-known antimicrobial material. These natural products inhibited the carboxyltransferase component of Escherichia coli acetyl-CoA carboxylase but had no effect on the activity of the biotin carboxylase component. The inhibition patterns indicated that these products bound to the biotin binding site of carboxyltransferase with trans-cinnamaldehyde having a Ki value of 3.8 ± 0.6 mM. The inhibition of carboxyltransferase by 4-hydroxy-3-methoxy-trans-cinnamaldehyde was analyzed with a new assay for this enzyme based on capillary electrophoresis. These results explain, in part, the antibacterial activity of this well-known antimicrobial material.