The aim of this in vitro study was to evaluate the wear and surface hardness of nine materials for conventional manufacturing, subtractive milling, and 3D printing of occlusal splints, as well as to evaluate the differences in wear and surface hardness between rigid and flexible 3D-printed occlusal splint materials. Two-body wear and Vickers hardness tests were performed. The vertical wear depth and Vickers hardness values were statistically analyzed. Vertical wear depth and surface hardness values were statistically significant among the investigated materials (p<0.05). The lowest vertical wear depth was observed for the heat-cured resin (27.5±2.4 μm), PMMA-based milled material (30.5±2.8 μm), and autopolymerizing resin (36.7±6.3 μm), with no statistical difference (p<0.05). Flexible 3D-printed and CAD-CAM milled polycarbonate-based splint materials displayed lower surface hardness and higher wear than the PMMA-based materials. PMMA-based splint materials displayed the most consistent surface hardness and wear resistance regardless of the manufacturing technology.