The effective management of marine species relies on a comprehensive understanding of how the environment can influence biodiversity and distributions. This study quantified the biodiversity of invertebrates across the phyla Arthropoda, Echinodermata, Mollusca and Porifera from 132 sampling stations along the coast of the Ningaloo Marine Park (NMP) and across depths ranging from 30 to 123 m, providing a broad assessment of the biodiversity of marine invertebrates in mesophotic habitats of the NMP. Benthic sleds were used to sample biodiversity and specimens identified to the highest taxonomic resolution. Single-beam and multibeam surveys of the seabed were performed to derive 58 spatial and bathymetric variables used to assess their relevance to the distributions of benthic diversity. A total of 787 taxa were identified across the four phyla, of which 51% were rare and sampled from a single station. Latitude and depth were significant in structuring the invertebrate diversity in the mesophotic, with Point Cloates notably identified as a significant geographical feature in partitioning diversity to the north and south of the NMP. Depth was more influential at structuring diversity in the northern section of the NMP due to the narrower Ningaloo Shelf, and southern localities including Turtles and Red Bluff were identified as biodiversity hotspots for benthic invertebrates. Importantly, comparisons of diversity within ‘no-take’ and ‘general use’ zones along the NMP indicate that sanctuary zones represent the diversity contained in the marine park, thus highlighting their importance in the protection and conservation of invertebrate diversity for the region.