The present study evaluated the parasite activity of aqueous extracts (AE) of neem leaves (Azadirachta indica) on parasitized Nile tilapia (Oreochromis niloticus). Concurrent infection with Dactylogyrus sp. and Centrocestus formosanus metacercariae was observed after clinical and parasitological investigations. A 240 Nile tilapia fish represented the four groups in the experimental design. They were used to assess the efficiency of aqueous extracts (long bath, 7 days) against parasite-infested fish: the group without A. indica extract (control, T1), T2 (1.0 g/L), T3 (2.0 g/L), and T4 (3.0 g/L). Results revealed that neem extract groups reduced the mean intensity of parasites (Dactylogyrus sp., C. formosanus) more than the control group partially at T4 (3.0 g/L). Fish exposed to neem leaf extracts showed significant downregulation of inflammatory and pro-inflammatory and immune genes (IL-1ß, IL-8; IL-10, Cox-2, lysozyme, C3, and HSP70) along with a significant increase in antioxidative genes (SOD and CAT) partially at T4 (3 g/L). Histological examinations of fish liver and gill tissues exposed to neem extract showed a return to normal histoarchitecture, especially at T4. However, antioxidant (CAT and GSH) activities increased in gill and liver tissues exposed to A. indica extract, especially at T4. Compared to control fish, the MDA levels decreased significantly in fish treated with A. indica extract, partially in the T4 group. Immune indices (complement 3, nitric oxide, and lysozyme activity) of infected fish treated with A. indica leaves extract displayed the highest value compared to the control one, especially at T4. The study assumed that an aqueous extract of A. indica inclusion at the rate of 3.0 g/L in a long-term bath is recommended to increase the resistance against parasitic infection and improve Nile tilapia’s health welfare.