High atomic number nanoparticles (NPs) have been shown to enhance the effects of radiation in vitro and in vivo. However, NPs are often observed to cluster together, leading to inhomogeneous distribution within the tissue and within cells themselves. The effect of this clustering on the capability of NPs to enhance radiation dose has not yet been fully investigated. In this Monte Carlo simulation study, the dependence of radio-enhancement on a separation parameter characterising NP clustering was investigated. A target water cube of side length 100 μm was simulated containing gold NPs constituting ~1% by mass. The NPs were placed in a cubic grid pattern and the separation distance between nanoparticles was varied. For NPs of 100 nm radius widely separated 2 μm apart, 91% of the total energy deposit was found to occur in the surrounding water, compared to only 56% when the NPs were moved closer together to 0.2 μm. The remaining energy deposit was absorbed by the NPs themselves. A similar trend was observed for NPs of radius 50 nm. The clustering effect was found to persist to greater separations for the larger NPs. The proportion of energy deposit in the available water of the target impacts the potential for cellular damage. Energy deposited within nanoparticles is unlikely to cause biological damage, as ionisations in the surrounding water are required to create radical oxygen species which then progress to cause the biological response to radiation. Clustering of nanoparticles is therefore expected to decrease their effectiveness for enhancing radiotherapy.