This study was designed to investigate, evaluate and compare the detoxifying efficiency of Penicillium roqueforti spores and Alpha-Tocopherol dietary supplementation in case of sublethal exposure of Nile tilapia (Oreochromis niloticus) to Lead. A total of 100 fish O. niloticus were divided into five equal groups. Group one, untreated control free aquarium water, was fed on basal diet lead acetate (ninety mg/L) was added to the aquarium water to the other four groups.  Group (2), lead acetate control positive fed on basal diet, group (3) fed on diet supplemented with (9×10² CFU/kg feed) of Penicillium roqueforti, group (4) fed on diet supplemented with Alpha-Tocopherol (300mg/kg feed), group (5) fed on diet supplemented with Penicillium roqueforti spores in addition to Alpha-Tocopherol as (9×10² CFU/kg feed and 300mg/kg feed) respectively. Nile tilapia fed at 3% body weight per day for 10 weeks.  Results of Pb intoxicated control positive group showed no characteristic clinical signs with presence of some postmortem and histopathological changes. Moreover serum analysis showed significant decreasing in growth hormone (GH), Calcium (Ca), phosphorus, Serum bactericidal activity and Serum lysozyme activity with increasing the mortality rates after challenging with Aeromonas hydrophila. Lead residues in blood, musculatures, gills, kidney and liver indicated that lowest Lead residue was recorded in musculatures but the highest residues were recorded in gills in all groups. Supplementation of Penicillium roqueforti spores and Alpha-Tocopherol improves the adverse effect of Lead in 3^rd, 4^th and 5^th groups. Best detoxification results were in 5^th group. It could be concluded that inclusions of 9×10² CFU /kg feed Penicillium roqueforti spores in addition to 300mg/kg feed  Alpha-Tocopherol in O.niloticus diets could reduce Lead adverse effects to the favor of fish health, immunity and minimizing the Pb residues specially in fish musculatures.