The present study evaluated the effect of aflatoxin B1 and Eimeria vaccine challenges and their interaction on intestinal morphology, applying the morphometric index “I See Inside” (ISI). Immune cellular response and broiler chicken performance were also studied. A total of 240 broiler chickens were divided in a 2 × 2 factorial arrangement with 4 treatments, T1: Control diet and no challenge (CON), T2: Aflatoxin B1 (AFLA), T3: Control diet and Eimeria challenge (COC), and T4: Aflatoxin B1 and Eimeria challenge (AFLA+COC). The mathematical morphometric index ISI was applied to evaluate macro and microscopic alterations. Samples of liver and jejunum were analyzed for macrophages, CD4+, and CD8+ cells counting by immunohistochemistry at 7, 14, and 21 d of age. Chickens challenged with Eimeria presented higher ISI of macroscopic alterations associated to Eimeria lesion at the medium small intestine, lower body weight gain (BWG) and feed intake (FI), and worse feed conversion ratio compared to non-challenged birds. Both Eimeria and aflatoxin challenges modulated the immune cells in the jejunum and liver, generally increasing the number of macrophages, CD4+, and CD8+ cells in relation to the control group. Birds from COC and COC+AFLA groups presented higher ISI histological score in the jejunum at 7 and 14 d of age compared to the CON and AFLA groups. The reduction of FI and BWG was correlated to high histological ISI and resulted in a high presence of immune cells in tissues, suggesting immune response demand. The histological ISI had statistical correlation to broiler performance.