The contamination of fish diets with aflatoxin B1 (AFB1) poses a significant hazard to the aquaculture sector. This study investigates the efficacy of supplementing Nile tilapia (Oreochromis niloticus) diets with garden cress (Lepidium sativum) seeds (GCS) to mitigate the adverse effects of AFB1. For this purpose, a factorial design was utilized, consisting of a 2×3 arrangement of treatments. This included two contamination levels of AFB1: (0 and 2 mg kg−1 diet). Within each contamination level, three dietary levels of GCS were used (0 %, 1 %, and 2 %). The study involved 270 healthy O. niloticus with an average weight of 25.14 ± 0.39 g (45 fish/group and 15 fish/replicate). The duration of the trial was 60 days. The study evaluated growth performance, hematological indices, hepatorenal function, immune and antioxidant indicators, histomorphology of organs, AFB1 residues in muscles, and fish resistance to Aeromonas hydrophila. The results indicated that GCS dietary supplementation significantly counteracted AFB1-induced growth retardation and reduced fish survival. Additionally, GCS effectively reversed the anemia, leukopenia, hypoproteinemia, hypoalbuminemia, and hypoglobulinemia that were produced by AFB1. Moreover, GCS dietary supplementation significantly suppressed the AFB1-induced increase in serum bilirubin, hepatic enzymes, kidney damage products, cortisol, and glucose. Additionally, the inclusion of GCS in the fish's diet effectively eliminated the accumulation of AFB1 in their muscles. Moreover, GCS supplementation demonstrated significant restoration of depleted antioxidant and innate immune components induced by AFB1. Furthermore, GCS significantly reduced the degenerative changes in renal, hepatic, splenic, and small intestine tissues caused by AFB1. Fish fed a GCS-fortified diet and exposed to AFB1 displayed restored disease resistance when challenged with A. hydrophila. In conclusion, supplementing Nile tilapia diets with GCS at 2 %, even in the presence the AFB1 contamination, significantly enhanced the fish growth, improved health, and increased disease resistance while reducing AFB1 residues, thereby benefiting consumer health.