Small-angle light scattering (SALS) was used to quantify the collagen fiber architecture of 12 bovine pericardium (BP) specimens overlying the right and left anterior surfaces of the heart. The collagen fiber architecture was described in terms of preferred collagen fiber directions, volume fractions (Vf), and degree of orientation. To explore the relationship between BP collagen architecture and mechanical anisotropy, biaxial mechanical tests were performed on two specimens chosen for their extremes of collagen fiber architecture. About 38% of the SALS test locations showed the occurrence of two distinct collagen fiber populations, with the primary population Vf typically 90%. Structurally, the left anterior specimens appeared to be the most homogeneous, with an overall preferred direction close to the circumferential direction of the heart. The right side specimens were inconsistent, with a broad range of fiber orientations. The direction of greatest stiffness was consistent with the underlying collagen fiber architecture. Overall, these results suggest that 1) the mechanical behavior of BP is quantitatively consistent with its collagen fiber architecture; 2) the variability of BP mechanical anisotropy is caused by variations in collagen fiber preferred directions; and 3) the left anterior side of the bovine pericardial sac might be a good material selection site for bioprosthesis fabrication.