Hot springs are the important natural sources of geothermally heated groundwater from the Earth's crust. Kuan-Tzu-Ling (KTL), Chung-Lun (CL) and Bao-Lai (BL) are well-known hot springs in southern Taiwan. Fluid and mud (sediments) samples were collected from the eruption points of three hot springs for detailed biogeochemical characterization. The fluid sample displays relatively high concentrations of Na(+) and Cl(-) compared with K(+), Mg(2+), Ca(2+), NO(2) (-), and SO(4) (2-), suggesting a possible marine origin. The concentrations of Fe, Cr, Mn, Ni, V and Zn were significantly higher in the mud sediments compared with fluids, whereas high concentrations of As, Ba, Cu, Se, Sr and Rb were observed in the fluids. This suggests that electronegative elements were released during sediment-water interactions. High As concentration in the fluids was observed to be associated with low redox (Eh) conditions. The FTIR spectra of the humic acid fractions of the sediments showed the presence of possible functional groups of secondary amines, ureas, urethanesm (amide), and silicon. The sulfate-reducing deltaproteobacterium 99% similar to Desulfovibrio psychrotolerans (GU329907) were rich in the CL hot spring while mesophilic, proteolytic, thiosulfate- and sulfur-reducing bacterium that 99% similar to Clostridium sulfidigenes (GU329908) were rich in the BL hot spring.