The data presented are related to the research article entitled "Evaluation of membrane fluidity of multidrug-resistant isolates of Escherichia coli and Staphylococcus aureus in presence and absence of antibiotics" (Bessa et al., 2018) [1]. This data article provides a dataset that includes emission spectra of Laurdan-labeled bacteria, namely of three Escherichia coli and three Staphylococcus aureus strains. Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) is a popular fluorescence probe used to assess membrane fluidity. Laurdan detects changes in membrane phase properties through its sensitivity to the polarity of the environment in the bilayer. Polarity changes are reflected by shifts in the Laurdan fluorescence emission spectrum that can be quantified by calculating the excitation generalized polarization (GPexc ).