Microdamage would be accumulated in bone due to high-intensity training or even normal daily activity, which may consequently cause fragility fracture or stress fracture. On the other hand, microdamage formation serves as a toughening mechanism in bone. However, the mechanisms that control microdamage initiation and accumulation in bone are still poorly understood. Our previous finite element model indicated that different interfacial properties between mineral and collagen in bone may lead to distinct patterns of microdamage accumulation. Therefore, the current study was designed to examine such prediction and to investigate the role of water and mineral–collagen interactions on microdamage accumulation in bone. To address these issues, 48 mice femurs were divided randomly into four groups. These groups were dehydrated or treated with perfluorotripropylamine (PFTA) or NaF solution to change water distribution and mineral–collagen interfacial bonding in bone. After three-point bending fatigue tests, the types of microdamage (i.e., linear microcracks or diffuse damage) formed in bone were compared between different groups. The results suggested that (1) bone tissues with strong mineral–collagen interfacial bonding facilitate the formation of linear microcraks, and (2) water has little contribution to the growth of microcracks.