The performance of nanoparticle assemblies with respect to various applications (e.g. sensors, catalysts, filtration membranes) depends on their microporosity. Here, the microcavities within the ligand matrix of superlattice films comprised of 1-dodecanethiol-stabilized gold nanoparticles (GNPs, core diameter ∼4 and ∼5.5 nm) were studied by positron annihilation lifetime spectroscopy (PALS). The superlattice composition, the size and spatial arrangement of the GNP cores were characterized by thermogravimetric analysis, transmission electron microscopy and grazing-incidence small-angle X-ray-scattering. From these data a structural model was derived to predict the sizes of the voids formed within the interstitial (tetrahedral and octahedral) sites of the superlattices. The comparison of the PALS-measured cavity sizes (0.50 to 0.74 nm) with the predicted void sizes of the interstitial sites (∼0.7 to ∼1.7 nm) and the free volume in solid dodecane (0.36 nm), previously measured by PALS, indicate that both types of cavities may contribute to the experimentally determined cavity sizes. However, the GNP core sizes had only a minor influence on the measured cavity size. Larger cavities with sizes corresponding to the voids (∼1.7 nm) expected within the octahedral sites of the superlattices comprised of ∼5.5 nm-sized GNP cores could not be detected. Assuming the intensities arising from these voids are measurable, this finding suggests that the octahedral sites are occupied by excess ligands trapped during film preparation. Apart from the voids predicted for the interstitial sites, the larger cavity sizes measured for the GNP superlattices compared to crystalline dodecane may result from some degree of disorder in the ligand arrangement.