Microbiology is at a turning point in its 120-year history. Widespread next-generation sequencing has revealed genetic complexity among bacteria that could hardly have been imagined by pioneers such as Pasteur, Escherich and Koch. This data cascade brings enormous potential to improve our understanding of individual bacterial cells and the genetic basis of phenotype variation. However, this revolution in data science cannot replace established microbiology practices, presenting the challenge of how to integrate these new techniques. Contrasting comparative and functional genomic approaches, we evoke molecular microbiology theory and established practice to present a conceptual framework and practical roadmap for next-generation microbiology.