Purpose <p><i>Acanthamoeba castellanii</i> is an important opportunistic human protozoal pathogen that can cause both skin, ocular and brain infections. Recent studies have established that brews and solvent extract (SE) of green tea (<i>Camellia sinensis</i>) can inhibit the growth and encystation of <i>A. castellanii</i>. Here we characterized those growth and encystation inhibitions.</p> Methods <p>Herein, we characterize of the morphological and chemical changes that occur in the trophozoites and the encysting stage of <i>A. castellanii</i> after exposure to <i>C. sinensis</i> SE and brew using Transmission electron microscopy (TEM), Fourier-transform infrared (FTIR) microspectroscopy and fluorescence-based assays.</p> Results <p>TEM showed ultrastructural changes in both <i>A. castellanii</i> stages. FTIR microspectroscopy revealed modifications of amide I and II band peaks in the <i>C. sinensis</i>-treated trophozoites, suggesting an inhibition of protein synthesis. Assessment of the nucleus integrity of trophozoites exposed to SE and brew revealed disruption of the nuclear membrane integrity, nuclear fragmentation, and chromatin degradation, and reduction in the quantity of DNA and RNA, indicating trophozoite death. These results are consistent with <i>C. sinensis</i> acting as a membrane-active anti-acanthamoebic, exhibiting amoebicidal activity against growing and encysting <i>A. castellanii</i>. This work underlines the importance of characterizing the effect of <i>C. sinensis</i> constituents, individually or in combinations, to clarify which ones are the primary components responsible for its action and the observed alterations in the structure and function of <i>A. castellanii</i>.</p> Conclusion <p>These results demonstrated that exposure to <i>C. sinensis</i> SE or brew alters the synthesis of protein, DNA, RNA and disrupts the cell wall integrity.</p>