Next-Generation Sequencing (NGS) technology allows to generate whole bacterial genome sequences in order to unequivocally characterize the strains to a highly discriminative resolution level. This methodology is used not only to study the bacterial clonality for tracing hospital outbreaks, but also to evaluate the presence of genes conferring antibiotic resistance or that encode for toxins. This makes NGS an appropriate technique that can be employed as ‘standard practice’ in hospitals, allowing to trace the circulation of “dangerous” strains. Here we describe the application of this technique in different clinical settings of suspected hospital-acquired infections sustained by relevant nosocomial pathogens. In particular, we analyze the more important genetic determinant and/or the mechanism conferring antibiotic resistance.