The cis-dihydroxylation of arenes by Rieske dearomatizing dioxygenases (RDDs) represents a powerful tool for the production of chiral precursors in organic synthesis. Here the substrate specificity of the RDD benzoate dioxygenase (BZDO) in R. eutropha B9 whole-cells was explored using quantitative nuclear magnetic resonance spectroscopy (q1H-NMR). The specific activity, specific carbon uptake, and regioselectivity of the dihydroxylation reaction were evaluated in resting cell cultures for a panel of seventeen mono-substituted benzoates. Two new substrates of this dioxygenase system were identified (2-methyl and 3-methoxybenzoic acid) and the corresponding cis-diol metabolites were characterized. Higher activities were observed for benzoates with smaller substituents, predominately at the 3-position. Elevated activities were also observed in substrates bearing greater partial charge at the C-2 position of the benzoate ring. The regioselectivity of the reaction was directly measured using q1H-NMR and found to have positive correlation with increasing substituent size. These results widen the pool of cis-diol metabolites available for synthetic applications and offer a window into the substrate traits that govern specificity for BZDO.