Cancer is an unmet global challenge causing a high socio-economic impact, worldwide. Every year, an increased number of patients become resistant to standard anti-cancer therapeutics, highlighting the need for novel targeted and patient-specific therapies. Recently, there has been an increased great deal of interest in the development of in vitro 3D tumor models. These emerging platforms can fill the gap between 2D cell studies and in vivo models. In fact, these novel benchtop tools can provide a deeper investigation of the molecular pathways underlying cancer development, in specifically designed 3D environments. Therefore, they could accelerate the discovery and validation of new anticancer therapies. These models can be fabricated using different categories of biomaterials, ranging from the synthetic to the natural origin. They can be further processed into different types of matrices/scaffolds, acting as extracellular matrix mimetics. The main types of biomaterials used for the generation of 3D in vitro tumor models and their recent applications are reviewed and discussed herein.