Bactericidal antibiotics kill by different mechanisms as a result of a specific interaction with their cellular targets. Over the past few years, alternative explanations for cidality have been proposed based on a postulated common pathway, depending on the intracellular production of reactive oxygen species. Detection of hydroxyl radicals relies on staining with specific fluorescent dyes that can penetrate the cell and are detected using flow cytometry. Flow cytometry has become an important tool in microbiology to study characteristics of individual cells within large heterogeneous cellular populations. We show here that Escherichia coli treated with different bactericidal antibiotics exhibits increased autofluorescence when analyzed by flow cytometry. We present evidence suggesting that this change in autofluorescence is caused by altered cell morphology upon antibiotic treatment. Consistent with this view, mutant cells that fail to elongate upon norfloxacin treatment show no increased auto-fluorescence response. Finally, we present data demonstrating that changes in autofluorescence can impact the results with fluorescent probes when using flow cytometry and confound the findings obtained with specific dyes. In summary, recent findings that correlate the exposure to cidal antibiotics with the production of reactive oxygen species need to be reconsidered in light of such changes in autofluorescence. Conclusive evidence for an increase of hydroxyl radicals after treatment with such drugs is still missing.