The aim of this research was to evaluate the influence of lettuce seed oil (LSO) on the performance, carcass yield, kidney and liver indices, immunity, lipid profile, and cecal microbiota of fattening chicks. A total of 200, 7-day-old Cobb-500 were distributed into 5 experimental groups; each group contained 5 replicates with 8 birds each. The first group 1) the basal diet (only); 2) the basal diet plus lettuce seed oil (0.50 mL/kg); 3) the basal diet plus lettuce seed oil (1.00 mL/kg); 4) the basal diet plus lettuce seed oil (1.50 mL/kg); and 5) the basal diet plus lettuce seed oil (2.00 mL/kg). No significant effect was observed on growth performance, carcass traits, or kidney function at any level of oil. But, liver function was significantly affected due to LSO levels. Serum lipid profiles (total cholesterol—TC, triglyceride—TG, low-density lipoprotein—LDL, and very low-density lipoprotein—VLDL) were significantly reduced by using LSO levels compared to the control group. Dietary LSO significantly increased immunological and antioxidant parameters, except for malondialdehyde-MDA, which was reduced. On the other hand, the cecal microbiota was significantly improved by LSO additives. It was concluded that the dietary supplementation of LSO had beneficial effects on liver and kidney functions, lipid profile, immunity, antioxidant parameters, and the bacteriology of fattening chicks.