Pathogenic bacteria encounter many stresses, from their environmental niches to colonization of their host. As unicellular organisms, a rapid and efficient response is crucial for their survival. Importantly, pathogenicity depends not only on the presence of virulence genes but also on how the bacterial chromosome is organized and regulated. Nucleoid-Associated Proteins (NAPs) are small, conserved proteins described as chromosome architects as their DNA-binding abilities allow them to remodel the nucleoid. Their direct impact on DNA transactions and their role as environmental sensors make NAPs key factors in adaptation, stress responses and virulence. During infection, they modulate foreign genetic material expression, transcription programs and interactions with host cells to resist harsh environments, establish colonization and counter host defenses. Here, we review the roles of NAPs following the infection process across various species, highlighting their essential and conserved functions at every step.