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How cells control their shape and size is a fundamental question of biology. In most bacteria, cell shape is imposed by the peptidoglycan (PG) polymeric meshwork that surrounds the cell. Thus, bacterial cell morphogenesis results from the coordinated action of the proteins assembling and degrading the PG shell. Remarkably, during steady-state growth, most bacteria maintain a defined shape along generations, suggesting that error-proof mechanisms tightly control the process. In the rod-shaped model for the Gram-positive bacterium Bacillus subtilis, the average cell length varies as a function of the growth rate, but the cell diameter remains constant throughout the cell cycle and across growth conditions. Here, in an attempt to shed light on the cellular circuits controlling bacterial cell width, we developed a screen to identify genetic determinants of cell width in B. subtilis. Using high-content screening (HCS) fluorescence microscopy and semiautomated measurement of single-cell dimensions, we screened a library of ∼4,000 single knockout mutants. We identified 13 mutations significantly altering cell diameter, in genes that belong to several functional groups. In particular, our results indicate that metabolism plays a major role in cell width control in B. subtilis. IMPORTANCE Bacterial shape is primarily dictated by the external cell wall, a vital structure that, as such, is the target of countless antibiotics. Our understanding of how bacteria synthesize and maintain this structure is therefore a cardinal question for both basic and applied research. Bacteria usually multiply from generation to generation while maintaining their progenies with rigorously identical shapes. This implies that the bacterial cells constantly monitor and maintain a set of parameters to ensure this perpetuation. Here, our study uses a large-scale microscopy approach to identify at the whole-genome level, in a model bacterium, the genes involved in the control of one of the most tightly controlled cellular parameters, the cell width.