This study aimed to evaluate the efficacy of cross-linked collagen membranes. Two types of collagen membranes were compared: a non-cross-linked collagen membrane (group A) and a cross-linked (group B) collagen membrane. In the in vitro study, the degradation rate in the presence of collagenase, the tear strength of the membranes, and the cytotoxicity of the cross-linked collagen membrane were evaluated. A total of 57 participants with cystic defects were randomized to undergo guided bone regeneration (GBR) using either membrane. Graft volume and new bone formation were measured by cone-beam computed tomography after 6 months of follow-up. In vitro findings revealed that the cross-linked collagen membrane retained more than 20% of its relative weight after 12 h. Meanwhile, the non-cross-linked collagen membrane exhibited complete degradation after 6 h. Clinically, no significant differences were observed between the groups in terms of graft resorption, new bone formation, and overall bone regeneration. These results indicate that cross-linking has comparable biocompatibility and enhances physical properties, including tear strength and resistance to degradation. However, clinical outcomes related to bone regeneration were not significantly different between cross-linked and non-cross-linked collagen membranes. Further research is warranted to determine the benefits of cross-linked collagen membranes in GBR procedures.