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Among various biomaterials used as scaffolds in tissue engineering, silk fibroin is a highly attractive material. A scaffold should be biocompatible and nontoxic, with optimal physical features and mechanical properties. For this reason, tissue-engineering approaches in regenerative medicine have focused on investigating the biocompatibility of possible biomaterials by analyzing cell–scaffold interaction properties. The aim of the present study was to examine the biocompatibility of silk fibroin as a film (two-dimensional [2D]) and a scaffold (three-dimensional [3D]) after being cellularized with human dental pulp stem cells (hDPSCs). Human dental pulp stem cells were isolated from healthy patients aged between 18 and 31 years. Further, silk fibroin-based 2D films and 3D scaffolds were prepared. Human dental pulp stem cells were directly seeded onto the biomaterial surfaces and their proliferation, adherence, and cell morphology were analyzed after 24, 120, and 168 hours. Additionally, the characteristics of the silk fibroin 2D films and 3D scaffolds before and after cell seeding were analyzed by scanning electron microscopy. After the initial 24 hours, silk fibroin-based 3D scaffolds displayed more adhered cells with a suitable fibroblastic morphology than those displayed on the 2D films. After longer culture times, hDPSCs proliferated sufficiently to cover the entire surface of the 3D silk fibroin scaffold, whereas the 2D films were only partially covered. Our results indicate the good in vitro biocompatibility of silk fibroin-based biomaterials, especially when 3D scaffolds rather than 2D films are used.