Abstract Cell membrane fluidity is an important phenotypic feature that regulates the diffusion, function and folding of transmembrane and membrane-associated proteins. It is particularly interesting to study it in bacteria as variations in membrane fluidity are known to affect fundamental cellular processes such as respiration, transport and antibiotic resistance. As such key parameter, membrane fluidity is regulated to adapt to environmental variations and stresses like temperature fluctuations or osmotic shocks. Membrane fluidity has been however scarcely studied quantitatively in bacterial cells, mostly because of the lack of available tools. Here, we developed an assay based on total internal reflection fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. We used this assay to quantify the fluidity of the cytoplasmic membrane of the Gram-positive model bacterium Bacillus subtilis in response to a cold shock, caused by a shift from 37°C to 20°C. In our experimental conditions, steady-state fluidity was recovered within 30 mins, and the steady-state fluidity at 20°C was about half of that at 37°C. Our minimally invasive assay opens up exciting perspectives and could be used to study a wide range of phenomena affecting the bacterial membrane, from disruption by antibiotics, antimicrobial peptides, or osmotic shocks. Significance Using fluorescence correlation spectroscopy (FCS) with total internal reflection fluorescence (TIRF) illumination, we measured the diffusion speed of fluorescent membrane markers as a readout for membrane fluidity of growing B. subtilis cells. Quantification of the effect of cold shock provided unique information about the dynamics of the plasma membrane of B. subtilis . The unprecedented capability of TIR-FCS to quantify membrane fluidity in living bacteria opens the door to a whole set of new studies that will shed light on the bacterial plasma membrane and its interactions with the environment.