This study aimed to explore the effects of different lipid sources on the performance, blood lipid parameters, immune system activity, and the expression of TNFα and TLR4 genes in broiler chickens. A total of 500 one-day-old male chicks of the ROSS 308 commercial strain were allocated into four treatment groups with five replicates each (each replicate comprised of 25 chickens), following a randomized design. The treatments were as follows: (1) a diet incorporating palm oil (PO, a source of saturated fatty acids); (2) a diet incorporating flaxseed oil (FO, a source of omega-3); (3) a diet incorporating soybean oil (SO, a source of omega-6); and (4) a diet incorporating olive oil (OO, a source of omega-9). According to the findings, the broiler chickens exhibited a significant increase in body weight gain (BWG) throughout the study when their diet consisted of unsaturated oils, as opposed to a diet including PO. Conversely, the feed conversion ratio (FCR) significantly decreased (P<0.01). The treatment with FO resulted in the highest percentage of lymphocytes and antibody titers against Newcastle and Gumboro diseases, showing a significant difference compared to the treatment with PO (P<0.01). Moreover, the relative expression of TNFα and TLR4 genes was the lowest following the FO treatment, indicating a significant decrease compared to the treatment with PO. Overall, the present findings demonstrated that incorporating omega-3 fatty acids into the diet was more effective in enhancing the growth performance, immune system, and health of broiler chickens.