Although acetate formation and tolerance are important criteria for various aspects of biotechnological process development, available studies on acetate tolerance in different species are disparate. We evaluate the response of eight bacterial strains, including two variants of Escherichia coli, two variants of Staphylococcus capitis, and one each of Acetobacter aceti, Gluconobacter suboxydans, Lactobacillus acetotolerans, and L. bulgaricus, to acetate challenges under identical conditions. Our findings were: (1) wild-type organisms of species that are considered tolerant of acetate perform only slightly better than E. coli in unadapted shaker cultures; (2) the ability to tolerate acetate is strongly dependent on the carbon source, and is, especially for E. coli, much greater on glycerol than on glucose; (3) respiration is not as important to acetate tolerance in E. coli and S. capitis as has been reported for the acetic acid bacteria; (4) S. capitis was the least affected by acetate under all conditions and grew at up to 44 g/l acetate without any preconditioning; and (5) qualitative high-throughput screening of growth characteristics can be achieved with relatively inexpensive multiwell plate readers.