The ecosystem and human health are being put at greater risk by the fungal pollution of water sources. In this study, the inactivation of the most common fungal isolate in water sources using ozone was reported. Six fungal isolates were isolated from Al-Azhar tap water, Al-Azhar wastewater, El-Menia canal water, and the El-Menia water treatment plant, where Aspergillus parasiticus (A. parasiticus) was the most common fungal isolate in all tested water samples. The fungi were morphologically identified, and A. parasiticus identification was confirmed using molecular identification. The variation of the dry weight of the isolated fungal species was reported upon exposure to different doses of ozone ranging from 10 μg/cc to 60 μg/cc, where the dry weight of A. parasiticus was the first fungal isolate that was significantly (P≤0.05) reduced upon using 10 μg/cc. The impact of using various doses of ozone for 5 minutes in metals levels on both Al-Azhar wastewater and El-Menia water treatment plant was compared versus using (50, 100, and 200 ppm) of chlorine, reflecting that using 10 μg/cc of ozone for 5 minutes could be applied as an effective dose in decreasing levels of metals. Furthermore, secretion of aflatoxins by A. parasiticus was dramatically decreased (P≤0.05) upon exposing the fungus to 10 mg/cc ozone for 5 minutes relative to untreated fungus. The present results revealed the possibility of using a low dose of ozone to decrease the growth of fungal pathogens in water and their possible secreted toxins.