Eruca sativa (ES) seeds are annual herbs belonging to the Brassicaceae family, widely grown in Mediterranean countries, such as Egypt, Italy, and Greece. The ES is rich in macronutrient components and phytochemical content, exhibiting potent antioxidant properties and functional properties for vital processes such as digestion and absorption of nutrients. Therefore, this research was conducted to evaluate the effects of dietary ES supplementation on laying performance, some blood parameters, and egg yolk cholesterol. A total of 300 Silver Sabahia strain hens, aged 26 weeks, were randomly distributed among four groups of five replicates, each replicate consisting of 15 hens. Chickens in group 1 served as a control and were fed the basal diet. Those in groups 2, 3, and 4 were fed basal diet supplemented with 1, 2, and 3% ESs, respectively. Productive performance traits, egg quality traits, hematological parameters, blood parameters, and yolk cholesterol profiles were performed throughout the study. The study lasted for 13 weeks (until week 39 of chickens' age). Results indicated that 3% ES supplementation had higher results on egg mass (35.68%), egg production (21.13%), and improved feed conversion ratio by 30.37%, compared to all groups. Furthermore, ESs supplementation positively affects the shell thickness and yolk color score compared to the control. Compared to the control, the highest significant blood hemoglobin and lymphocytes were recorded in the groups supplemented with 2% and 3% of ESs. The ES inclusion at a higher level (3%) in the diet of laying hens led to significantly enhanced serum high-density lipoprotein and total antioxidant capacity, while reducing cholesterol, low-density lipoprotein, and malondialdehyde levels, compared to the control diet. Serum calcium, tri-iodothyronine, and alkaline phosphatase levels increased significantly in response to 3% ES treatment, while liver enzymes decreased significantly compared to the control diet. Notably, the addition of 2% and 3% ESs to the hens’ ration resulted in reduced egg cholesterol content, which is desirable for consumers seeking healthier dietary choices. Finally, adding 3% ESs to hens’ diet improves productive performance, egg quality traits, hematological parameters, blood parameters, and yolk cholesterol profile.