Ehlers–Danlos syndrome (EDS) is a genetic disease leading to abnormalities in mechanical properties of different tissues. Here we quantify corneal biomechanical properties in an adult classic EDS mouse model using two different measurement approaches suited for murine corneal mechanical characterization and relate differences to stromal structure using Second Harmonic Generation (SHG) microscopy. Quasi-static Optical Coherence Elastography (OCE) was conducted non-invasively during ambient pressure modulation by − 3 mmHg. 2D-extensometry measurements was conducted invasively consisting of a pre-conditioning cycle, a stress-relaxation test and a rupture test. In a total of 28 eyes from a Col5a1+/− mouse model and wild-type C57BL/6 littermates (wt), Col5a1+/− corneas were thinner when compared to wt, (125 ± 11 vs 148 ± 10 μm, respectively, p < 0.001). Short-term elastic modulus was significantly increased in OCE (506 ± 88 vs 430 ± 103 kPa, p = 0.023), and the same trend was observed in 2D-extensometry (30.7 ± 12.1 kPa vs 21.5 ± 5.7, p = 0.057). In contrast, in stress relaxation tests, Col5a1+/− corneas experienced a stronger relaxation (55% vs 50%, p = 0.01). SHG microscopy showed differences in forward and backward scattered signal indicating abnormal collagen fibrils in Col5a1+/− corneas. We propose that disturbed collagen fibril structure in Col5a1+/− corneas affects the viscoelastic properties. Results presented here support clinical findings, in which thin corneas with global ultrastructural alterations maintain a normal corneal shape.