Flow-through fractures dominate the movement of fluids in a variety of natural as well as engineered subsurface systems. Microbial activities in fractured rock impact subsurface energy recovery, storage, and waste disposal. It has been recognized that understanding how the contrasting permeability between fracture and matrix interacts with microbial metabolism under thermal and hydrological gradients is key to effective utilization of the subsurface, yet such studies are sparse. Microorganisms mediate the production of hydrogen sulfide (also known as souring) in oil-bearing geological formations. We conducted a comprehensive experimental study of a novel 2D fractured rock system to understand these complex interactions and demonstrated how biofilm development can impact fracture flow, which subsequently feedbacks to moderate sulfidogenesis. Elevated temperature relevant to reservoir conditions interacted with the injection of cold fluid and formed a thermal gradient away from fractures, creating thermal niches for microbial activities in the fractured rock. Results showed that while fracture flows were dominant in the beginning, with time, growth of the biofilm in the fractures reduced permeability, effectively moderating the initial fracture-matrix contrast, and limited microbial accessibility to nutrients and subsequent reactions rates.