Acanthamoeba castellanii is a widespread unicellular eukaryote found in diverse environments, including tap water, soil, and swimming pools. It is responsible for severe infections, such as Acanthamoeba keratitis and granulomatous amebic encephalitis, particularly in individuals with immunocompromisation. The ability of protozoans to form dormant and persistent cysts complicates treatment, as current therapies are ineffective against cyst stages and suffer from poor specificity and side effects. Nitroxoline, a quinoline derivative with well-established antibacterial, antifungal, and antiviral properties, is a promising therapeutic candidate. This study aimed to elucidate cellular signalling events that counteract the effects of nitroxoline. In this study, nitroxoline significantly reduced the viability of A. castellanii trophozoites in a dose- and time-dependent manner, inducing morphological changes and apoptosis. Transcriptomic analysis revealed substantial alterations in gene expression, including enrichment of metabolic pathways, DNA damage responses, and iron ion binding. Nitroxoline treatment upregulated genes involved in DNA repair and oxidative stress response while regulating genes in the methionine and cysteine cycles. It also decreased the mitochondrial membrane potential, H&sub2;S production, and total iron amount in A. castellanii. Bioinformatic analyses and molecular docking studies suggest direct interactions between nitroxoline and several A. castellanii proteins. Our research provides a comprehensive molecular map of the response of A. castellanii to nitroxoline, revealing significant changes in gene expression related to the stress response and metabolic pathways. These findings underscore the potential of nitroxoline as a potent anti-Acanthamoeba agent, offering new insights into its mechanism of action and paving the way for effective combinational therapeutic strategies.