Photodynamic therapy (PDT) is a potentially non-invasive therapeutic strategy that involves the use of a photosensitizing agent molecule capable to absorb light at a specific wavelength and to transfer the absorbed energy to generate singlet oxygen. This oxidizing agent reacts with double bonds of membrane phospholipids and sterols present in biological membranes causing damages and/or cell permeability. With the aim to analyze and to determine the effectiveness of possible photosensitizing agents, in this work we evaluate the in vitro amoebicidal effect of nine cosmetic dyes, 1–9, belonging to the xanthene, chlorophyll, aminoanthraquinone, and pyrene group of chromophores, against Acanthamoeba castellanii Neff. Red 22 (2, eosin Y) and red 28 (4) were the most effective photosensitizers to inhibit cell growth, with 2 showing the highest growth inhibition when it is activated by white light. The study of their mechanisms of action reveals that red 22 (2) increases membrane permeability and induces chromatin condensation of parasites, and could be used in combined therapies with currently used drugs.