Abstract <p>Here we examined how drug-to-lipid ratio affects the fine structure of anionic liposomes with dipalmitoyl phosphatidylcholine and cardiolipin loaded with levofloxacin following with investigation of molecular details of complex formation with mannosylated chitosan. Drug loading leads to reduce of acyl chain mobility in the bilayer and redistribution of carbonyl and phosphate groups on the hydration degree, suggesting bilayer structural changes. Higher drug-to-lipid ratio provides more pronounced changes. The effects become more significant upon heating, mostly because of slowing down of phase transition. Complex formation with mannosylated chitosan mostly mitigates effects. Thus, altering drug-lipid-ratio affects the structure of anionic levofloxacin-loaded liposomes coated with mannosylated chitosan, affecting their stability and therapeutic capacity.</p>