In ovo injection of nano‐selenium (Se) produced by lactic acid bacteria (LAB–nano‐Se) was investigated on the hatchability, immune responses and the histopathological alterations in hatched chicks. The eggs (18 day age) were injected with 0.5 ml of 0.9% NaCl (normal saline, NS), while the control group was kept without injection. In the third, fourth and fifth groups, the eggs were injected with 0.5 ml of NS and LAB–nano‐Se at 10, 20 and 30 μg/egg. The results revealed improved growth performance in groups injected with LAB–nano‐Se when compared to the control treatment. The highest final weight and weight gain were noticed in 20 μg LAB–nano‐Se/egg group (p < .05). The feed conversion ratio was reduced in all treated groups when compared to the control group (p < .05). Groups injected with LAB–nano‐Se showed enhanced hatchability of the whole incubated eggs (p < .05). Total lipids and cholesterol levels were decreased significantly in groups treated with LAB–nano‐Se at 10 and 20 μg/egg when compared to the non‐treated group. At the same time, globulin was increased by LAB–nano‐Se in ovo injection. Furthermore, the total antioxidant capacity, catalase, glutathione peroxidase, superoxide dismutase increased in groups treated with LAB–nano‐Se at 10 and 20 μg/egg with insignificant (p > .05) differences with those treated with LAB–nano‐Se at 30 μg/egg using in ovo injection technique. Also, higher total blood protein and phagocytosis were significantly observed in groups treated with at 10, 20 and 30 μg LAB–nano‐Se/egg. The histopathological images of hatched chicks revealed that nano‐Se presented normal effects on liver and kidney tissues and restored the parameters as mentioned earlier. To conclude, LAB–nano‐Se exhibited beneficial effects in hatched chicks through improving immune and antioxidant activities as well as histopathological effects by using in ovo technique.