Three-dimensional collagen matrices of porcine origin are being used as substitutes for soft tissue grafts in periodontal plastic surgery in search of aesthetic and natural results. This in vitro study aimed to compare Fibro-Gide® (GeistlichBiomaterials) and Mucoderm® (BotissBiomaterials) matrices during the initial phase of soft tissue formation. For this purpose, samples of 5 × 5 mm were obtained, and then human fibroblasts were plated on them. After 24, 48 and 72 h, cell viability was assessed using an MTT assay, and the secretion of type I collagen, MMP-2, TIMP-1 and TIMP-2 was analyzed by ELISA immunoassay. The control group (C) consisted of cells plated on polystyrene without the matrices. The morphology of the surfaces was also examined using scanning electron microscopy (SEM), as was the average roughness (Ra) of the samples by a profilometer. Topographic analysis revealed that roughness was significantly higher on Mucoderm® than on Fibro-Gide® (p < 0.05). Human gingival fibroblasts plated on both matrices showed similar results for cell viability as the cells cultured on C (p > 0.05). The synthesis of type I collagen, MMP-2 and TIMP-1 were significantly higher from cells plated on Fibro-Gide® than on Mucoderm®, in all time points (p < 0.05). Furthermore, at 24 and 48 h, TIMP-2 secretion was also significantly higher on Fibro-Gide® than on Mucoderm® (p < 0.05). Based on these results, it is possible to conclude that even though both matrices demonstrated cell viability, Fibro-Gide® induced an increase in type I collagen, MMP-2 and TIMP-1 and TIMP-2.