Microorganisms that generate biofuels from lignocellulosic biomass face a multifaceted environment of inhibitory by-products from pretreatment processes, pentose and hexose sugars, and the biofuels themselves. In order to create a more cost-effective and viable fermentation process, these variables must be addressed when selecting and engineering an organism for biofuel production. One significant effect of biofuels on the organism is the deterioration of the cell membrane, which protects the organism and is crucial to cell viability. The influence of cell type was investigated when various species of yeast, archaea, and bacteria were grown in the presence of potential biofuels, such as ethanol, butanol, and isobutanol. Lipids were extracted from the respective organisms and used in lipid composition and fluidity studies. Membrane composition was determined when these organisms were exposed to an amount of biofuel that reduced growth rate. Using fluorescence anisotropy, membrane fluidity was measured of liposomes from each organism as a function of the biofuel concentration. The specific growth rates of each organism declined and the membrane fluidity increased as alcohol concentrations increased. The unique lipid composition between organisms contributed to their varying degrees of tolerance in each potential biofuel.