Purpose <p>In this study, the bioprospecting of prokaryotes associated with the sulfur (S) cycle was carried out using culture-dependent and culture-independent methods in the acid-thermal crater lake “El Chichón” in Mexico.&#xa0;</p> Methods <p> In the first approach, prokaryotic communities from environmental samples (sediments, water, and sediment vents) were surveyed using 16S rRNA gene amplicon metabarcoding. Then, sediment samples were cultured under anaerobic and acid conditions at 50°C using diverse media, C-sources, and S-electron acceptors (SO₄^2-, S₂O₃^2- and S⁰), and then also characterized.&#xa0;</p> Results <p> The crater-lake presented thermo-acidic conditions (pH 2.7–4.0, temperatures reaching up to 85°C) and we found S-species such as sulfate (365–741 ppm) and sulfide (0.79–2.96 ppm). The highest concentrations of both compounds were detected in sediment vents. The environmental samples showed the dominance of the genera<i>Thermoplasma</i>,<i>Acidianus</i>,<i>Acidithiobacillus</i>,<i>Mesoaciditoga</i>,<i>Ammonifex</i>,<i>Thiomonas</i> and <i>Desulfurella</i>, all related to S-cycle redox processes. On the other hand, the anaerobic enrichments showed a dominance of <i>Desulfofundulus</i>genus using sulfate as an electron acceptor, <i>Mesoaciditoga</i>in elemental sulfur, and <i>Desulfurella</i>with thiosulfate. The functional annotation of prokaryotic taxa showed a dominance of sulfur metabolisms.&#xa0;</p> Conclusion <p> Our study provided detailed information about the dominance of potential functions of the sulfur biogeochemical cycle and new insights for understanding the adaptation of the complex microbiota and their ecological importance in a dynamic crater-lake.</p>