Seventeen human clinical isolates representing four species of Desulfovibrio were characterized using 16S rRNA gene sequences and tests for catalase, indole, nitrate, bile, urease, formate-fumarate stimulation, desulfoviridin, motility, and hydrogen sulfide production, plus susceptibility to antimicrobial agents. Eighty additional strains representing 10 phenotypically similar genera (Bilophila, Selenomonas, Capnocytophaga, Campylobacter, Bacteroides, Sutterella, Anaerobiospirillum, Dialister, Veillonella, and Mobiluncus) were included for comparison. All Desulfovibrio species produced H2S and were desulfoviridin positive, and all Desulfovibrio species except D. piger were motile. The four Desulfovibrio species could be distinguished from each other using tests for catalase, indole, nitrate, urease, and growth on bile, with the following results (positive [+], negative [-], growth [G], and no growth [NG]): for D. piger, -, -, -, -, and G, respectively; for D. fairfieldensis, +, -, +, -, and G, respectively; for D. desulfuricans, -, -, +, +, and NG, respectively; and for D. vulgaris, -, +, -, -, and G, respectively. Resistance to the 10-microg colistin disk separated the Desulfovibrio species from most of the other genera, which were usually susceptible. These simple tests were useful for characterizing the Desulfovibrio species and differentiating them from other phenotypically similar genera.