High concentrations of indole are known to be toxic to cells due to perturbations in membrane potential. Here, we report for the first time a transcriptome analysis of a soil model bacterium, Pseudomonas putida KT2440, under indole treatment. We demonstrated that 47 genes are differentially expressed, including 11 genes involved in the tricarboxylic acid cycle (TCA cycle) and 12 genes involved in chaperone and protease functions (hslV, hslU, htpG, grpE, dnaK, ibpA, groEL, groES, clpB, lon-1, lon-2, and hflk). Mutant analysis supported the observation that protease genes including hslU are essential for the indole resistance of Pseudomonas strains. Subsequent biochemical analyses have shown that indole increases the NADH/NAD+ ratio and decreases the adenosine triphosphate (ATP) concentration inside cells, due to membrane perturbation and higher expression of TCA cycle genes in the presence of indole. This energy reduction leads to a reduction in cell size and an enhancement of biofilm formation in P. putida. The observed upregulation in many chaperones and proteases led us to speculate that protein folding might be inhibited by indole treatment. Interestingly, our in vitro protein-refolding assay using malate dehydrogenase with purified GroEL/GroES demonstrated that indole interferes with protein folding. Taken together, our data provide new evidence that indole causes toxicity to P. putida by inhibiting cellular energy production and protein folding.