The spider family Dictynidae is a diverse and widespread group of spiders, found globally in both terrestrial and semi-aquatic environments. Recent work has suggested that a substantial amount of undescribed biodiversity exists in the Global South, particularly in regions of sub-Saharan Africa and Madagascar. To investigate and describe this biodiversity, specimens of dictynid spiders from Madagascar in the California Academy of Sciences collections were examined and species hypotheses were formulated based upon morphology.  In this study, we tested these morphological hypotheses using genetic data as an independent line of evidence. To accomplish this, we sequenced three genes (CO1, 12s, H3) for 39 museum specimens and inferred a phylogeny of these dictynids, including potentially related species from mainland Africa. We found that most of the morphospecies hypotheses were supported by the genetic data, increasing confidence in the species delimitations made. Furthermore, using phylogenetic analyses, we identified the African species most closely related to these new species in Madagascar, and found potential evidence that there was a single dispersal event of this lineage into Madagascar, with subsequent speciation on the island. This study illustrates the vastly unknown endemic diversity of Madagascar, and further supports evidence of the utility of museum collections and low-cost Sanger sequencing approaches, for confirming morphological species hypotheses.