Zymomonas mobilis ZM4 is a model ethanogenic bacterium due to its productivity, high level of ethanol tolerance and its ability to be genetically manipulated. In this study, we developed a Phenotype MicroArray™ (PM) protocol to profile nearly 2,000 Z. mobilis cellular phenotypes. The PM panels included assays for carbon, nitrogen, phosphorus and sulfur source utilization, nutrient stimulation, pH and osmotic stresses, and chemical sensitivities with 240 inhibitory chemicals. The PM analysis gave an overview of the Z. mobilis physiological characteristics, such as the limited C-source (fructose and glucose) utilization, which was consistent with literature reports. For nitrogen metabolism, it utilized ammonia and, for single amino acids it preferred aspartate, asparagine, glutamine, and glutamate. Likewise, for peptide utilization, it preferred peptides with aspartate, asparagine, glutamate, glutamine, and glycine, although others were used more slowly. Z. mobilis appeared to use a diverse array of P-sources with the exception of pyrophosphate and tripolyphosphate. The assays suggested Z. mobilis uses both inorganic and organic compounds as S-sources. No stimulation by nutrients was detected, however, there was evidence of partial inhibition by purines and pyrimidines, NAD, and deferoxamine. Z. mobilis was relatively resistant to acid pH, tolerating a pH down to about 4.0. It also tolerated phosphate, sulfate, and nitrate but was rather sensitive to chloride and nitrite. Z. mobilis showed resistance to a large number of diverse chemicals that inhibit most bacteria. The information obtained provides fundamental insights into the physiology of Z. mobilis that may assist future metabolic engineering endeavors.