Cable bacteria are filamentous sulfur-oxidizing microorganisms that couple the reduction of oxygen or nitrate in surface sediments with the oxidation of free sulfide in deeper sediments by transferring electrons across centimeter scale distances. The distribution and activities of cable bacteria in freshwater sediments are still poorly understood, especially the impact of cable bacteria on sulfur cycling. The goal of this study was to investigate electrogenic sulfide oxidation associated with cable bacteria in laboratory microcosm incubations of freshwater sediments using microsensor technology, 16S full-length rRNA sequencing, and fluorescence in situ hybridization (FISH) microscopy. Their activity was characterized by a pH maximum of 8.56 in the oxic zone and the formation of a 13.7 ± 0.6 mm wide suboxic zone after 25 days of incubation. Full-length 16S rRNA gene sequences related to cable bacteria were recovered from the sediments and exhibited 93.3%-99.4% nucleotide (nt) similarities with those from other reported freshwater cable bacteria, indicating that new species of cable bacteria were present in the sediments. FISH analysis indicated that cable bacteria density increased with time, reaching a maximum of 95.48 m cm on day 50. The cells grew downwards to 40 mm but were mainly concentrated on the top 0-20 mm of sediment. The cable bacteria continuously consumed HS in deeper layers and oxidized sulfide into sulfate in the 0-20 mm surface layers, thereby affecting the sulfur cycling within sediments. These findings provide new evidence for the existence of higher diversity of cable bacteria in freshwater sediments than previously known.