Klebsiella pneumoniae is a ubiquitous bacterial pathogen associated with nosocomial infections with the emergence of hypervirulent strains driving the appearance of community infections. Certain virulence factors of K. pneumoniae are well characterized; however, the acquisition of new genetic material can introduce novel virulence traits, constantly changing our understanding of infection. Bacterial pathogenesis reflects growth and survival within the host environment, evading and responding to immune cells, and interacting with other microbes that may be present. In this thesis research, I describe the application of mass spectrometry-based proteomics to identify novel aspects influencing diverse areas of K. pneumoniae pathogenesis. I profiled the impact of metal (i.e., iron and zinc) availability on the proteome of K. pneumoniae, offering insight into nutritional immunity during infection. Here, I identified an uncharacterized protein, ChaB, with novel connections to zinc homeostasis and explored its relevance to virulence factor production. Additionally, I defined a putative role in the regulation of iron homeostasis towards Lon protease within the extracellular environment. Moreover, I investigated the interactions driving infection between K. pneumoniae and primary BALB/c macrophages to define promising infection-associated K. pneumoniae proteins and characterized their roles in bacterial growth, morphology, and virulence. Importantly, mutations to three genes encoding uncharacterized proteins displayed reduced virulence during in vitro and in vivo infection models and I discovered unique bacteria-host interacting partners that offer new insight into K. pneumoniae pathogenesis. Finally, I profiled a K. pneumoniae infection in the presence of the opportunistic fungal pathogen, Cryptococcus neoformans, to detect species-specific regulation and defense mechanisms within the lung environment during infection and I explored proteins produced by each organism. Proteomic profiling revealed suppression of C. neoformans in the presence of K. pneumoniae showcasing the complexity of microbial interactions and proposing novel strategies to combat dual infections. The implementation of quantitative proteomics analyses in these key areas of K. pneumoniae pathogenesis provided a global overview of bacterial adaptation and survival within distinct environments, along with interactions driving disease. Overall, this thesis research contributes to our knowledge of K. pneumoniae pathogenesis and identifies proteins as putative novel targets for anti-virulence discovery.