Time window of antibiotic administration is a critical but long‐neglected point in the treatment of bacterial infection, as unnecessary prolonged antibiotics are increasingly causing catastrophic drug‐resistance. Here, a second near‐infrared (NIR‐II) fluorescence imaging strategy based on lead sulfide quantum dots (PbS QDs) is presented to dynamically monitor bacterial infection in vivo in a real‐time manner. The prepared PbS QDs not only provide a low detection limit (104 CFU mL−1) of four typical bacteria strains in vitro but also show a particularly high labeling efficiency with Escherichia coli (E. coli). The NIR‐II in vivo imaging results reveal that the number of invading bacteria first decreases after post‐injection, then increases from 1 d to 1 week and drop again over time in infected mouse models. Meanwhile, there is a simultaneous variation of dendritic cells, neutrophils, macrophages, and CD8+ T lymphocytes against bacterial infection at the same time points. Notably, the infected mouse self‐heals eventually without antibiotic treatment, as a robust immune system can successfully prevent further health deterioration. The NIR‐II imaging approach enables real‐time monitoring of bacterial infection in vivo, thus facilitating spatiotemporal deciphering of time window for antibiotic treatment.