Streptococcus pneumoniae (the pneumococcus) is the etiologic agent of community-acquired pneumonia and invasive pneumococcal diseases such as septicemia and bacterial meningitis. The increasing antibiotic resistance and the suboptimal efficacy or limited serotype coverage of currently available vaccines urgently requires novel approaches in exploring new antimicrobials, therapeutic intervention strategies and vaccines. The current vaccine development strategies rely on the hypothesis that surface-exposed proteins, which are essential for pneumococcal virulence, are the most suitable candidates for future protein-based vaccines. Since virulence is closely linked with bacterial fitness, the potential of a pathogen to colonize and infect the host depends further on its physiology. This review summarizes the application of genome-wide techniques and their exploitation to decipher fundamental insights into bacterial factors associated with fitness, metabolism and virulence, leading to the discovery of vaccine candidates or antimicrobials.