A well-defined silica nanoparticle model system was developed to study the effect of the size and structure of aggregates on their membranolytic activity. The aggregates were stable and characterized using transmission electron microscopy, dynamic light scattering, nitrogen adsorption, small-angle X-ray scattering, infrared spectroscopy, and electron paramagnetic resonance. Human red blood cells were used for assessing the membranolytic activity of aggregates. We found a decreasing hemolytic activity for increasing hydrodynamic diameter of the nanoparticle aggregates, in contrast to trends observed for isolated particles. We propose here a qualitative model that considers the fractal structure of the aggregates and its influence on membrane deformation to explain these observations. The open structure of the aggregates means that only a limited number of primary particles, from which the aggregates are built up, are in contact with the cell membrane. The adhesion energy is thus expected to decrease resulting in an overall lowered driving force for membrane deformation. Hence, the hemolytic activity of aggregates, following an excessive deformation of the cell membrane, decreases as the aggregate size increases. Our results indicate that the aggregate size and structure determine the hemolytic activity of silica nanoparticle aggregates.