Essential oils of Citrus sinensis and Citrus latifolia have shown biological functions as antiseptics, anti-inflammatories, antioxidants, antifungal and antimutagenic, so the evaluation of their antibacterial capacity, by themselves or in combination with standard antibiotics, presents an alternative for infection treatment. Flow cytometry opens the door for the design of faster and more accurate measurement of antibacterial activity. We use a SYTO9/PI staining system on E. coli ATCC 25922 to determine antibacterial activity by counting live and dead cells through flow cytometry. We found that dual staining showed highly variable results due to wavelength overlapping and instead we used fluorochrome individual staining that highly correlated with viable counts. Chloramphenicol and cefotaxime treatments did not present a dose-response behavior, rendered diffuse readings and/or gave filament formation on fluorescence microscopy. Amikacin was a better comparison standard because it presented a dose-response behavior. Essential oils had low antibacterial activity as compared to amikacin, with a maximum of 10% and 20% for C. latifolia and C. sinensis, respectively. Combinations of essential oils with antibiotic resulted in an unforeseen strong inhibition of amikacin activity. Although a low antibacterial activity was found, a series of standardization steps are proposed for antibacterial activity measurement by flow cytometry.