Over the past decade regenerative branches of dentistry have taken on more and more importance, resulting in the development of performing scaffold materials. These should induce cell adhesion, support, and guide the tissues’ growth. Among the developed materials, we can include resorbable or non-membranes. The purpose of this study was to investigate the proliferation abilities and the attachment of human periodontal ligament fibroblasts (HPLIFs) over two bovine pericardium membranes with different thicknesses, 0.2 mm and 0.4 mm, respectively. These membranes have been decellularized by the manufacturer, preserving the three-dimensional collagen’s structure. The HPLFs were cultured in standard conditions and exposed to the tested materials. XTT was performed to assess cell proliferation, while light microscopy (LM) and scanning electron microscopy (SEM) observations assessed fibroblast morphology at different times (T1, T2, and T3). Proliferation assays have shown a statistically significant difference in growth at T1 (p < 0.05) in the cells cultured with a thicker membrane compared to the thinner one. LM analysis showed healthy fibroblasts in contact with the membranes, appearing larger and with a polygonal shape. SEM observation demonstrated thickening of the fibroblasts which continued to adhere to the membrane’s surface, with enlarged polygonal shape and developed filipodia and lamellipodia. These results showed a similar cell behavior over the two bovine pericardium membranes, demonstrating a cellular migration along and within the layers of the membrane, binding with membrane fibers by means of filopodial extensions. Knowledge of the effects of the collagen membranes derived from bovine pericardium on cellular behavior will help clinicians choose the type of scaffolds according to the required clinical situation.