Prokaryotic diversities of 12 geothermal hot springs located in Northern, Central and Southern Tunisia were investigated by culture-based and molecular approaches. Enrichment cultures for both aerobic and anaerobic microorganisms were successfully obtained at temperatures ranging from 50 to 75 C. Fourteen strains including four novel species were cultivated and assigned to the phyla Firmicutes (9), Thermotogae (2), Betaproteobacteria (1), Synergistetes (1) and Bacteroidetes (1). Archaeal or universal oligonucleotide primer sets were used to generate 16S rRNA gene libraries. Representative groups included Proteobacteria, Firmicutes, Deinococcus-Thermus, Thermotogae, Synergistetes, Bacteroidetes, Aquificae, Chloroflexi, candidate division OP9 in addition to other yet unclassified strains. The archaeal library showed a low diversity of clone sequences belonging to the phyla Euryarchaeota and Crenarchaeota. Furthermore, we confirmed the occurrence of sulfate reducers and methanogens by amplification and sequencing of dissimilatory sulfite reductase (dsrAB) and methyl coenzyme M reductase a-subunit (mcrA) genes. Altogether, we discuss the diverse prokaryotic communities arising from the 12 geothermal hot springs studied and relate these findings.