ABSTRACTShigellaare Gram-negative bacterial pathogens responsible for bacillary dysentery (also called shigellosis). The absence of a licensed vaccine and widespread emergence of antibiotic resistance has led the World Health Organisation (WHO) to highlightShigellaas a priority pathogen requiring urgent attention. Several infection models have been useful to explore theShigellainfection process; yet, we still lack information regarding events taking placein vivo. Here, using aShigella-zebrafish infection model and high-content microscopy, we developed an automated microscopy workflow to non-invasively study fluorescently labelled bacteria and neutrophilsin vivo. We applied our workflow to antibiotic-treated zebrafish, and demonstrate that antibiotics reduce bacterial burden and not neutrophil recruitment to the hindbrain ventricle. We discovered that nalidixic acid (a bactericidal antibiotic) can work with leukocytes in an additive manner to controlShigella flexneriinfection and can also restrict dissemination ofShigella sonneifrom the hindbrain ventricle. We envision that our automated microscopy workflow, applied here to study the interactions betweenShigellaand neutrophils as well as antibiotic efficacy in zebrafish, can be useful to innovate treatments for infection control in humans.