Amphipod crustaceans are a major group of invertebrates that predominantly occur in groundwater ecosystems. Eoniphargus is a mesogammarid genus with only two known species from the groundwater systems of the Japanese archipelago and Korean Peninsula. However, there is a dearth of taxonomic studies on this genus, and the species diversity within Eoniphargus is unclear. Here, we describe two new species, E. iwataorum sp. nov. and E. toriii sp. nov., collected from the interstitial waters in Tochigi and Shizuoka Prefectures in the Japanese archipelago. These two new species are distinguished from their congeners by the following features: head, urosomite 3, first and second antennae, mandibles, and maxilla 1. Eoniphargus kojimai is redescribed here based on material collected near the type locality. Molecular phylogenetic analyses based on the nuclear 28S rRNA and mitochondrial COI genes revealed that E. kojimai is sister to E. iwataorum sp. nov. In this study, we also briefly discuss the phylogenetic relationships of Mesogammaridae based on the molecular phylogenetic analyses.