Using autogenous grafts in mucogingival surgeries is related to postoperative morbidity and limited tissue availability, and thus xenogeneic matrices are increasingly used. This in vitro study evaluated the influence of xenogeneic collagen matrix thickness on cell adhesion, morphology, viability, proliferation, and matrix degradation. Matrices were divided into three groups: SLC: single layer of Lumina Coat, as commercially available (2-mm thickness); DLC: double layer of SLC (Lumina Coat); and MG: single layer of Mucograft, as commercially available (4-mm thickness). SEM was used to evaluate the matrix surface topographies. To evaluate the cell viability, proliferation, adhesion, and morphology, human gingival fibroblasts (HGF) and stem cells from human exfoliated deciduous teeth (SHED) were used. Cell viability was evaluated through MTS colorimetric method evaluating HGF and SHED on days 1, 3, and 7. Cell proliferation was assessed by PicoGreen assay, evaluating HGF and SHED on days 3 and 7. Sample degradation was evaluated on days 1, 3, 7, 14, 21, 28, and 35. All groups were biocompatible for HGF and SHED, showing viabilities > 70% on days 1, 3, and 7. DCL promoted HGF viabilities similar to MG (P = .2828) and the highest SHED viability (P < .0001) on day 1. DLC also demonstrated HGF and SHED proliferations higher than the positive control (MG; P < .05) on day 7. SLC was completely degraded on day 14, while DLC and MG presented 48.41% and 20.52% of their initial mass, respectively, on day 35. Increasing the matrix thickness improved HGF and SHED viability and proliferation and prevented early matrix degradation. DLC demonstrated better results than SLC and MG concerning matrix degradation and HGF and SHED viability and proliferation.