To precisely determine the mechanical properties of bone, the effects of preservation on these properties need to be minimized. It seems probable that prolonged exposure to formalin or saline will have some effect on the mechanical properties of bone. This study investigated the effects of formalin preservation on the fracture toughness of bovine femoral cortical bone (Haversian bone and plexiform bone) including the eluted bone mineral concentrations in the preservative solution. The periods of formalin preservation were 30 and 150 days. To confirm anisotropic properties, two different specimens were used: circumferential and radial specimens with initial fissures in the lateral cross section were examined. The fracture toughness tests were performed using three point bending specimens at a crosshead speed of 1 or 20 mm/min. It was confirmed that the fracture toughness of the bone decreased, with the highest figure showing a 30% reduction in fracture toughness when formalin preservation was employed. The fracture toughness reduction rate was higher in the plexiform bone sample at a crosshead speed of 20 mm/min. The eluted Ca concentration from the specimen in saline was higher than that in formalin. Thus, preservative aqueous solutions such as saline were shown to significantly dissolve bone mineral.