The Barberton Greenstone Belt (BGB), South Africa, consists of volcano-sedimentary and sedimentary successions deposited between 3.5 and 3.2 Ga, which are subdivided into three Groups: the Onverwacht, Fig Tree, and Moodies. After deposition, the Barberton Greenstone Belt underwent relatively low-grade tectonothermal events, enabling estimation of surface environmental events and biological evolution in the Middle Archean. Isotopic studies suggest the activity of methanogen, sulfate-reducing bacteria, and photosynthetic bacteria at 3.4 Ga. Microbial dissimilatory iron reduction (DIR) is also considered to be one of the earliest expressions of metabolism on Earth, but the isotopic signal of microbial DIR is still uncertain in the Archean. We performed in-situ iron isotope analyses of individual pyrites in sedimentary rocks from the BGB, using a femtosecond laser ablation multi-collector ICP-MS technique (fs-LA-MC-ICP-MS) to find isotopic evidence for the microbial activity. We obtained a large variation in δ56Fe values, ranging from -1.84 to + 3.79 ‰. Most of the δ56Fe values of pyrites from the Hooggenoeg Complex show positive values, whereas those from the Noisy Complex and subsequent sedimentary sequences show a wide variation from negative to positive δ56Fe values. One of the main differences between these Complexes is their depositional depth. The Hooggenoeg Complex was probably deposited in a deep-ocean environment, whereas the Noisy Complex was in shallow water. The negative δ56Fe values of pyrite grains in the Noisy Complex, Onverwacht Group indicate the occurrence of partial reduction caused by microbial DIR in a Middle Archean shallow sea.