Abdominal aortic aneurysms (AAA) are common and potentially life-threatening aortic dilatations, due to the effect of hemodynamic changes on the aortic wall. Previous research has shown a potential pathophysiological role for increased macroscopic aneurysmal wall stiffness; however, not investigating micromechanical stiffness. We aimed to compile a new protocol to examine micromechanical live aortic stiffness (elastic moduli), correlated to histological findings with quantitative immunofluorescence (QIF). Live AAA biopsies (n = 7) and non-dilated aortas (controls; n = 3) were sectioned. Local elastic moduli of aortic intima, media and adventitia were analysed in the direction towards the lumen and vice versa with nanoindentation. Smooth muscle cells (SMC), collagen and fibroblasts were examined using QIF. Nanoindentation of AAA vs. controls demonstrated a 4-fold decrease in elastic moduli (p = 0.022) for layers combined and a 26-fold decrease (p = 0.017) for media-to-intima direction. QIF of AAA vs. controls revealed a 4-, 3- and 6-fold decrease of SMC, collagen and fibroblasts, respectively (p = 0.036). Correlations were found between bidirectional intima and media measurements (ρ = 0.661, p = 0.038) and all QIF analyses (ρ = 0.857–0.905, p = 0.002–0.007). We present a novel protocol to analyse microscopic elastic moduli in live aortic tissues using nanoindentation. Hence, our preliminary findings of decreased elastic moduli and altered wall composition warrant further microscopic stiffness investigation to potentially clarify AAA pathophysiology and to explore potential treatment by wall strengthening. Statement of Significance: Although extensive research on the pathophysiology of dilated abdominal aortas (aneurysms) has been performed, the exact underlying pathways are still largely unclear. Previously, the macroscopic stiffness of the pathologic and healthy aortic wall has been studied. This study however, for the first time, studied the microscopic stiffness changes in live tissue of dilated and non-dilated abdominal aortas. This new protocol provides a device to analyse the alterations on cellular level within their microenvironment, whereas previous studies studied the aorta as a whole. Outcomes of these measurements might help to better understand the underlying origin of the incidence and progression of aneurysms and other aortic diseases.