Non-growing cells can function as whole-cell biocatalysts for hydrogen (H₂) production, a process that has recently drawn much attention. In order to evaluate their potential as whole-cell biocatalysts, we compared the H₂-production capability of four Enterobacteriaceae strains (Citrobacter amalonaticus Y19, Escherichia coli K-12 MG1655, Escherichia coli DJT135, and Enterobacter aerogenes) under growing and non-growing conditions. We evaluated their H₂-production activity at varying temperatures (25–45°C) and pH conditions (6.0–8.0) using glucose or formate as the carbon source. Under growing conditions with 10mM glucose as a substrate, E. aerogenes exhibited the highest H₂-production activity (17.0±0.2μmolH₂mgcell⁻¹h⁻¹) among the four strains, but the final H₂ yield was similar (1.7–1.8molH₂mol⁻¹ glucose) in all four strains. H₂ production in the four strains proceeded through a formate-dependent pathway that involved the formate hydrogen lyase (FHL) complex. Under non-growing conditions with 20mM formate as a substrate, we obtained high H₂-production activities, in the range of 95.5–195.2μmolH₂mgcell⁻¹h⁻¹, with E. coli DJT135 exhibiting the highest activity (195.2μmolH₂mg⁻¹h⁻¹) at pH 6.0 and 45°C. In contrast, using glucose as the carbon substrate in non-growing cell experiments greatly reduced the H₂-production activity to 6.1–7.7μmolH₂mgcell⁻¹h⁻¹. This study indicated that formate is a better substrate than glucose for H₂ production by non-growing cells, and that the H₂-production performance among the strains did not vary significantly, with the exception of E. coli K-12 MG1655.