Studying the variables that affect the membrane fusion mechanism of enveloped viruses is important for developing new strategies to combat viral infections. We analysed the effects of lipid vesicle cholesterol content on membrane fusion that is facilitated by infectious salmon anaemia virus (ISAV) fusion peptides. Lipid mixing assays were performed to study membrane fusion in large unilamellar vesicles (LUV) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and cholesterol. Lipid mixing (%) increased more over time when 0.2 µm LUV contained no cholesterol or when the LUV membranes contained 15 mol% cholesterol. The secondary structure of the ISAV fusion peptides consistently remained a β-sheet both in water and in the presence of vesicles. Additionally, the dissociation constant (Kd) between the peptides and the lipid vesicles was obtained with different cholesterol contents. In the tests performed with lipid vesicles (0.2 µm or 0.4 µm LUV), cholesterol was found to influence membrane fusion that was facilitated by ISAV fusion peptides; however, the peptides studied did not require cholesterol in their membranes to facilitate membrane fusion in the smallest lipid vesicles (0.2 µm LUV).