Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of important human pathogens. However, evidence from previous antimicrobial screenings, in silico analysis, and experimental target evaluation suggests that the number of novel bacterial broad-spectrum targets might be severely limited. This is because of the poor conservation of genes among diverse bacterial pathogens, partial functional redundancy and nutrient-rich host environments. Remaining opportunities under these circumstances include the development of narrow-spectrum antibiotics against specific pathogens and the exploration of target combinations.