Abiotic stresses, such as heat, can drive protein misfolding and aggregation, leading to inhibition of cellular function and ultimately cell death. Unexpectedly, a thermotolerant Escherichia coli was identified from a pool of antibiotic resistant RNA polymerase β subunit (rpoB) mutants. This stress tolerant phenotype was characterized through exposure to high temperature and ethanol. After 30-minute exposure of cells to 55°C or 25% ethanol, the mutant displayed 100 times greater viability than the wild-type, indicating that the rpoB mutation may have broadly affected the cellular environment to reduce protein misfolding and/or prevent protein aggregation. To further test this hypothesis, we examined thermotolerance of cells lacking heat shock chaperone DnaJ (Hsp40), which is a cochaperone of one of the most abundant and conserved chaperones, DnaK (Hsp70). The deletion of dnaJ led to severe growth defects in the wild-type, namely a slower growth rate and extreme filamentation at 42°C. The severity of the growth defects increased after additionally deleting DnaJ analog, CbpA. However, these defects were significantly ameliorated by the rpoB mutation. Finally, the rpoB mutant was found to be minimally affected by the simultaneous depletion of DnaK and DnaJ compared to the wild-type, which failed to form single colonies at 37°C and 42°C. Based on these observations, it is proposed that the rpoB mutant’s robust thermotolerant phenotype results from a cellular environment protective against protein aggregation or improper folding. The folding environment of the rpoB mutants should be further examined to elucidate the mechanism by which both antibiotic resistance and thermotolerance can be conferred.