The integrity of the bacterial cytoplasmic membrane is critical in maintaining the viability of cells and their metabolic functions, particularly under stress. Bacteria actively adjust membrane fluidity through changes in lipid composition in response to variations in temperature, pressure, ion concentrations, pH, nutrient availability, and xenobiotics. Fluorescence polarization methods are valuable for measuring bacterial cytoplasmic membrane fluidity. In this review we discuss the mechanisms of bacterial membrane adaptations and present data from research using 1,6-diphenyl-1,3,5-hexatirene (DPH) as a measure of membrane fluidity and phase transitions. We illustrate the range of fluidity in viable cells, extracted membranes, and liposomes under optimal and stressed physiological conditions.