A hydrogenotrophic, mesophilic methanogen designated as strain T53BJ was isolated from anaerobic digester continuously fed with agro-industrial waste. Cells were stained Gram negative, spiral shaped, motile and found resistant to cell lysis by SDS and hypotonic solution. Growth occurred with optimum (i) temperature of 30 °C (20–40 °C) (ii) pH of 7.0 (6.5–8.5), in the presence of 0.0–0.2 M NaCl with a doubling time of 73.43 h and utilized only hydrogen and carbon dioxide (H2:CO2; 4:1) as a sole source of carbon and energy. The phenetic and phylogenetic analysis based on 16S rRNA and mcrA gene sequences revealed close affiliation of T53BJ strain to the genus Methanospirillum spp. This is the first report about isolation of hydrogenotrophic Methanospirillum spp. from anaerobic digester fed with fruit waste, press mud and neem seed cake, suggesting its potential use for bioaugmentation of anaerobic reactors or upgrading biogas/ syngas to methane.