One borehole was installed in the Blackfoot Disease (BFD) area to collect organic-carbon and metal-rich sediments from various depths. Carbon- and metal-rich groundwaters were also collected to quantify arsenic and humic substances from different parts of the BFD area in Chianan Plain. UV–visible spectroscopy was performed to detect the presence of iron in humic substances. Fourier Transform Infrared (FTIR) spectra showed that the functional groups such as COO−, CO, OH, and CC (aromatic ring) were present in humic substances of southwestern Taiwan, and they act as chelating agents to bind with trace elements in sediments. Nuclear magnetic resonance (NMR) spectroscopy also suggests that signals peaking at 30, 58, 68, 101.22, 168.23 and 210ppm were assigned to the presence of aliphatic carbon, N-alkayl C, O-alkayl C, anomeric carbon, carboxyl carbon, and ketonic carbon of functional groups, respectively. The binding nature of arsenic with humic substances was also characterized using FTIR microscopy. FTIR spectra of laboratory-synthesized As–Fe–humic substance (HS) complexes were compared to those of Fe–HS complex and humic substances in order to demonstrate the presence of binary Fe–HS and ternary As–Fe–HS complexes in the aquifer. Organometallic complex (humic substances in combination with arsenic) in groundwater of southwestern Taiwan may cause BFD.