The heterotrophic, aerobic, facultatively anaerobic, catalase- and oxidase-positive, non-motile strain MWH-PoolGreenA3(T), isolated from a rock pool filled with freshwater, was characterized. The strain grew on NSY medium over a NaCl range of 0.0-0.3 % (w/v). Whole-cell fatty acids were dominated by C(16 : 1)ω7c (feature 3), C(18 : 1)ω7c and straight-chain C(16 : 0); furthermore, the components C(12 : 0) and C(14 : 0) 2-OH were present. The DNA G+C content was 48.3 mol%. Phylogenetic analysis as well as strong similarities in phenotypic and chemotaxonomic traits indicated the affiliation with the genus Polynucleobacter. 16S rRNA gene similarity values with the three described species of the genus Polynucleobacter ranged from 96.7 to 97.8 %. DNA-DNA hybridization experiments did not reveal that the strain belongs to a previously described species of the genus Polynucleobacter. The strain can be discriminated from previously established species of the genus Polynucleobacter by chemotaxonomic and phenotypic traits. The bacterium possesses a free-living lifestyle and represents a group of planktonic freshwater bacteria occurring with high cell numbers in many freshwater lakes. Based on the phylogeny revealed and the chemotaxonomic and phenotypic differences from previously described species of the genus Polynucleobacter, we propose to establish the novel species Polynucleobacter acidiphobus sp. nov. with the type strain MWH-PoolGreenA3(T) ( = DSM 21994(T) = CIP 110079(T)).