Nitrobacter species play a key role in nitrogen cycling; however, their physiological adaptations and genomic diversity remain largely underexplored. This study characterizes Nitrobacter vulgaris strain MLSD-S22, isolated from a nitrate- and heavy-metal-contaminated subsurface, revealing unexpected genomic and metabolic traits. Higher nitrite and oxygen affinities compared to previous reports indicate that some Nitrobacter are more competitive in low-substrate environments than previously believed. The presence of a complete nitrous oxide (N2O) reduction operon highlights the genomic potential related to N2O reduction within this genus; however, we did not observe N2O reduction under the conditions tested. By expanding Nitrobacter ecophysiology, this work provides a foundation for future research on the metabolic flexibility of Nitrobacter and their contributions to nitrogen transformations in diverse environments.