Natural Killer (NK) cells are cytotoxic lymphocytes and key mediators of innate immunity, essential for combating viral infections and cancer. Notably, they exhibit immunological memory, generating a stronger response upon re-exposure to the same stimulus. While NK cell memory holds promise for infection control, its role in bacterial infections remains poorly understood. Previously, we demonstrated that Streptococcus pneumoniae induces long-term, specific, and protective NK cell memory. In this study, we performed single-cell RNA-seq to uncover how NK cells respond to S. pneumoniae infection. Our findings reveal that challenged Memory (cMemory) NK cells undergo transcriptional reprogramming following S. pneumoniae infection and have a differential transcriptional response upon reinfection. In addition, we identified distinct cMemory NK cell subpopulations, with responding cMemory NK cells displaying a general enhanced activation, proliferation, and cytotoxic activity. These findings support a novel role for NK cells in the context of bacterial infections, thereby opening avenues for harnessing the potential of innate immune memory for therapeutic applications.