The current study was conducted to evaluate the effect of different levels of dietary supplementation of herbal essential oil and byproduct mixture ( Thymus vulgaris, Mentha piperita,Rosmarinus offisinalis and Anethum graveolens ) and different dietary fat sources on performance, egg quality, immunological and histological responses of laying hens. In this study, 240 hen layers were randomly distributed among two of 12 experimental treatments with 4 replicates of 5 birds each. At the first trial, dietary treatment consisted of three levels of herbal essential oil mixture (0, 100 and 200 mg/kg) and two sources of fat (fish oil and soybean oil), and in the second experiment, treatments included three levels of herbal byproducts blend (0, 2.5 and 5%) and two sources of fat (fish oil and soybean oil) that fed during a 80 d feeding trial including 10 days for adaptation period and 70 days as main recording period. Performance parameters including feed intake, feed conversion ratio, egg production percentage, egg weight, egg mass and also egg quality were calculated on d 35, and 70 of experiment. To investigate of immunological response, sheep red blood cells (SRBC) were injected in two times with 10 d interval; thereby, hens were bled 6 and 12 d after the first and second injection, respectively. Moreover, Newcastle disease virus was injected and then sera were taken 6 d after injection. In addition, differential leukocyte counts were evaluated at the end of experiment. On d 70 of experiment, two birds of each cage were exsanguinated to evaluate the relative weight of internal organs, and also to sample ileal content and jejunal morphology. The results showed that adding fish oil significantly increased spleen weigh (P 0.01), liver malondialdehyde (MDA) concentration (P 0.05), egg color index (P 0.05), egg mass (P 0.05), number and diameter of laminaproperia lymphoid follicles (P 0.05) and also led to a reduction in serum ALP enzyme activity (P 0.01), serum cholesterol and triglyceride concentration (P 0.01), yolk cholesterol (P 0.01), hepatic fat (P 0.01), heterophil to lymphocyte ratio (P 0.05) and villus height (P=0.08). Moreover, herbal essential oil mixture also led to a reduction in blood cholesterol and triglyceride concentration (P 0.01), enzyme activities of SGPT and SGOT (P 0.01), the relative weight of liver (P 0.01), heterophil to lymphocyte ratio (P 0.05), intestinal pathogen population (P 0.01) and hepatic and serum MDA concentration (P 0.05). Also, it caused an improvement in antibody titer against SRBC and Newcastle disease virus (P 0.01), villus height to crypt depth ratio and diameter of laminaproperia lymphoid follicles (P 0.01) as well as egg shell hardness and thickness (P 0.01). Additionally, administration of different levels of herbal byproducts only improved antibody titer against SRBC (P 0.05), number and diameter of laminaproperia lymphoid follicles and goblet cell (P 0.01) and egg shell quality (P 0.05) and also ileal Escherichia coli population (P 0.01). In general, results indicated that dietary inclusion of fish oil with 200 mg/kg essential oil mixture led to the best immunological and intestinal histological responses, a modulation in serum lipid profile and also egg mass index. Furthermore, humoral immunity, bacterial population and egg shell quality were remarkably affected by supplementation of herbal byproduct mixture. Keywords: Thymus vulgaris, Mentha piperita, Rosmarinus offisinalis, Anethum graveolens, fish oil, performance, egg quality, laying hens.