Abstract Trichodina species can cause extensive fish mortality in an aquaculture system, and extensive use of commercial drugs affects the environment and health. The bacterial zinc nanoparticles (BZN) synthesized using Bacillus subtilis MS20 could be a suitable alternative to antiparasitic drugs. This work evaluates the antiparasitic properties of BZN against Trichodina heterodentata in Nile tilapia (Oreochromis niloticus). Furthermore, it explores BZN's effects on fish health, including growth performance, immunity, and microbial load. The BZN were 45nm in size and negatively charged at -23.6 meV. A total of 180 fish were divided into six groups. G1-G4 were non-trichodina-infected groups fed a diet supplemented with 0, 100, 200, and 500 mg/kg of BZN, the G5 was Trichodina-infected fish, and G6 was Trichodina-infected fish and treated with BZN (500 mg/kg). Fish were carefully observed for clinical alterations and mortalities. Blood and tissue samples were collected from different groups. Survival rates were 100% in the groups (G1-G4), 56.67% (G5), and 88 % (G6). The Trichodina-challenged group showed bad performance, especially in the survival rate (56 %) and body weight gain, lower erythrogram (Hb, RBCs, however, higher PCV%, which may indicate the inflammation in fish tissue), significant hypoglycemia, higher ALP activity and ammonia but lower protein content and significant increases were observed in serum amyloid A (SAA) and interleukin-6 (IL-6) values. Applying BZN 500 mg/kg to the challenged group maintains the parameters to control levels and alleviates the effect of Trichodina species infection. The histopathology of liver tissues supported the blood biochemistry results. The Trichodina-challenged fish showed extensive vacuolar degeneration of hepatocytes and massive necrosis of the pancreas hyperplastic proliferation of the epithelial lining of the secondary lamellae; meanwhile, BZN-treated groups showed normal liver and gill tissue structure. The BZN treatment significantly reduced the total bacterial count by 31-45% and Aeromonas count by 30-44%. Finally, it is concluded that using BZN 500 mg/kg significantly mitigates the Trichodina heterodentata infection in Nile Tilapia (Oreochromis niloticus) and enhances fish health.