Sediment accumulation in water storage tanks may protect microorganisms from disinfectant exposure, causing the degradation of water quality. However, microbial activity and disinfectant penetration within water storage sediment remain largely uncharacterized. This study evaluated the penetration of monochloramine and free chlorine into a 2 cm (20000 μm) deep drinking water storage tank sediment using microelectrodes. The sediment was successively exposed to monochloramine for 4 months, free chlorine for 2 months, and monochloramine for 2 months. Temporal monochloramine, free chlorine, dissolved oxygen (DO), pH, ammonium, nitrite, and nitrate profiles were acquired using microelectrodes. The results showed that complete monochloramine or free chlorine penetration was not observed. Likewise, DO never fully penetrated the sediment, progressing inward with time to a maximum depth of 10000 μm and indicating microbial activity persisted over the entire 8 months. Decreasing ammonium and increasing nitrate concentrations, with minimal nitrite accumulation, further demonstrated microbial activity and indicated complete sediment nitrification. There were measurable levels of ammonium, nitrite, and nitrate during free chlorine application, and nitrification activity gradually resumed upon a switch back to monochloramine. These findings suggest that the periodic removal of sediment from drinking water storage facilities is desirable to remove potentially protected environments for microorganisms.