Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health. Display omitted •A Drosophila resource of mutants affecting adult neural development and maintenance•Essential fly genes with multiple human homologs are often associated with disease•Variants in human homologs of these genes are associated with Mendelian disease•ANKLE2 is associated with small brain size and microcephaly in fly and man Combining datasets from a Drosophila resource of mutants with neurodevelopmental or -degenerative phenotypes with human exomes of patients with rare Mendelian disorders provides new insights into human neurodevelopmental diseases.