The redox conditions strongly affect redox kinetics, hydrochemical composition and microbial reactions under river bank filtration (RBF). For investigating the redox conditions, zonation factors, and microbial responses in a typical RBF with high Fe2+ and Mn2+ contents, three wells were established 20 m apart perpendicular to the south bank of the Songhua River with a constant flow rate of 400 m3/h. Redox zonation was identified from the redox sequences, redox products, reactions identified and seasonality. High-throughput sequencing, principal coordinates analysis, and redundancy analysis were used to identify the microbial responses to redox zonation. The results indicated that the RBF was stabilized after 21 d of water extraction with a 51%, 36% and 21% decrease of Fe2+, Mn2+ and NH4+ contents, respectively. Feature redox indices varied with strong regularity in the horizontal and vertical filtration directions, and the filtration area could be divided into O2/NO3−, Fe/Mn, and SO42− reduction zones. Seasonality resulted in markedly fluctuation of Fe2+ and Mn2+ contents in groundwater. Pseudomonas, Flavobacterium, and Limnobacter were the dominant bacteria in the O2/NO3−, Fe/Mn, and SO42− reduction zones, respectively. DO explained 21.1% of microbial variability in the fluctuation zone, while Mn2+ and Fe2+ explained 27.7%, and 26.2%, respectively, of microbial variability in the saturated zone.