Hopanoids are a class of membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against the solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in this bacterium has limited their further functional analysis. Due to the polyploidy (>50 genome copies per cell) of Z. mobilis, we found that disruptions of essential hopanoid biosynthesis (hpn) genes act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid polar head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their head group composition, contributes to fitness at low pH. Spectroscopic analysis of bacterial-derived liposomes showed that hopanoids protect against several ethanol-driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter-lipid hydrogen bonding interactions to stabilize bacterial membranes during solvent stress.