| Summary: | <i>Acanthamoeba</i> Keratitis (AK) can lead to substantial vision loss and morbidity among contact lens wearers. Misdiagnosis or delayed diagnosis is a major factor contributing to poor outcomes of AK. This study aimed to assess the effect of two antibiotics and one anaesthetic drug used in the diagnosis and nonspecific management of keratitis on the autofluorescence patterns of <i>Acanthamoeba</i> and two common bacteria that may also cause keratitis. <i>Acanthamoeba castellanii</i> ATCC 30868, <i>Pseudomonas aeruginosa</i> ATCC 9027, and <i>Staphylococcus aureus</i> ATCC 6538 were grown then diluted in either PBS (bacteria) or ¼ strength Ringer’s solution (<i>Acanthamoeba</i>) to give final concentrations of 0.1 OD at 660 nm or 10<sup>4</sup> cells/mL. Cells were then treated with ciprofloxacin, tetracycline, tetracaine, or no treatment (naïve). Excitation–emission matrices (EEMs) were collected for each sample with excitation at 270–500 nm with increments in 5 nm steps and emission at 280–700 nm at 2 nm steps using a Fluoromax-4 spectrometer. The data were analysed using MATLAB software to produce smoothed color-coded images of the samples tested. <i>Acanthamoeba</i> exhibited a distinctive fluorescence pattern compared to bacteria. The addition of antibiotics and anaesthetic had variable effects on autofluorescence. Tetracaine altered the fluorescence of all three microorganisms, whereas tetracycline did not show any effect on the fluorescence. Ciprofloxacin produced changes to the fluorescence pattern for the bacteria, but not <i>Acanthamoeba</i>. Fluorescence spectroscopy was able to differentiate <i>Acanthamoeba</i> from <i>P. aeruginosa and S. aureus</i> in vitro. There is a need for further assessment of the fluorescence pattern for different strains of <i>Acanthamoeba</i> and bacteria. Additionally, analysis of the effects of anti-amoebic drugs on the fluorescence pattern of <i>Acanthamoeba</i> and bacteria would be prudent before in vivo testing of the fluorescence diagnostic approach in the animal models.
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