Discovering virulence factors of pathogenic bacteria is a key in understanding pathogenesis and for identification of targets for novel drugs and design of new vaccines. Comparative genomics, transcriptomics, and proteomics have become the popular tools in discovering the virulence factors in bacterial pathogens, such as Neisseria meningitidis, Yersinia pestis, Mycobacterium tuberculosis, and Staphylococcus aureus. In addition, proteomics has been employed successfully in the study of the mechanism of post-translationally modified proteins of bacterial pathogens. Once the putative virulence factors are identified by genomics and/or proteomics, their functions and mechanisms can be further investigated by phenotypic analyses including mutagenesis and biochemical methods and/or structural biology. Combination of these techniques will accelerate the developments of therapeutic drugs and vaccines in combating bacterial diseases.