Nucleariid amoebae (Opisthokonta) have been known since the nineteenth century but their diversity and evolutionary history remain poorly understood. To overcome this limitation, we have obtained genomic and transcriptomic data from threeNuclearia, twoPompholyxophrysand oneLithocollaspecies using traditional culturing and single-cell genome (SCG) and single-cell transcriptome amplification methods. The phylogeny of the complete 18S rRNA sequences ofPompholyxophrysandLithocollaconfirmed their suggested evolutionary relatedness to nucleariid amoebae, although with moderate support for internal splits. SCG amplification techniques also led to the identification of probable bacterial endosymbionts belonging to Chlamydiales and Rickettsiales inPompholyxophrys. To improve the phylogenetic framework of nucleariids, we carried out phylogenomic analyses based on two datasets of, respectively, 264 conserved proteins and 74 single-copy protein domains. We obtained full support for the monophyly of the nucleariid amoebae, which comprise two major clades: (i)Parvularia–Fonticulaand (ii)Nucleariawith the scaled generaPompholyxophrysandLithocolla. Based on these findings, the evolution of some traits of the earliest-diverging lineage of Holomycota can be inferred. Our results suggest that the last common ancestor of nucleariids was a freshwater, bacterivorous, non-flagellated filose and mucilaginous amoeba. From the ancestor, two groups evolved to reach smaller (Parvularia–Fonticula) and larger (Nucleariaand related scaled genera) cell sizes, leading to different ecological specialization. TheLithocolla+Pompholyxophrysclade developed exogenous or endogenous cell coverings from aNuclearia-like ancestor.This article is part of a discussion meeting issue ‘Single cell ecology’.