To compare mechanical, optical, and physical properties of denture base materials fabricated with various 3D printing systems to reference milled and conventionally heat-processed denture base materials. Specimens of denture base materials were either 3D-printed (DLP in-office printer, CLIP laboratory printer, or material jetting laboratory printer), milled, or heat processed. 3-point bend flexural strength testing was performed after 50 hours of water storage following 1hour of drying (dry testing) or in 37°C water (wet testing). Fracture toughness was performed with a notched beam specimen after 7 days of water storage and tested dry. The translucency parameter was measured with 2mm thick specimens. Stain resistance was measured as color change following 14 days of storage in 37°C coffee. Water sorption was measured following 7 days of storage in 37°C distilled water. For dry testing, all but one of the 3D-printed materials attained higher or equivalent flexural strength as the reference materials. For wet testing, all 3D-printed materials attained higher or equivalent strength as the reference materials and dry-tested materials. For 3D-printed materials, wet testing increased displacement before fracture whereas it decreased displacement for the reference materials. Only two of the 3D-printed materials had similar fracture toughness as the reference materials. One of the 3D-printed materials was more translucent and one was more opaque than the reference materials. Only one of the 3D-printed materials absorbed more water than the reference materials. 3D-printed denture base materials have mostly equivalent mechanical, optical, and physical properties to conventional and milled denture base materials.