Polarization-dependent second harmonic generation (PSHG) microscopy is used as an innovative, high-resolution, non-destructive, and label-free diagnostic imaging tool to elucidate biological issues with high significance. In the present study, information on the structure and directionality of collagen fibers in equine pericardium tissue was collected using PSHG imaging measurements. In an effort to acquire precise results, three different mathematical models (cylindrical, trigonal, and general) were applied to the analysis of the recorded PSHG datasets. A factor called the “ratio parameter” was calculated to provide quantitative information. The implementation of the trigonal symmetry model to the recorded data led to the extraction of improved results compared with the application of the widely used cylindrical symmetry model. The best outcome was achieved through the application of the general model that does not include any kind of symmetry for the data processing. Our findings suggest that the trigonal symmetry model is preferable for the analysis of the PSHG datasets acquired from the collagenous tissues compared with the cylindrical model approach although an increased computational time is required.