Sports-related dental trauma remains a risk for children and adolescents. Although mouthguards provide protection, up to 25% of dentoalveolar injuries can occur with a mouthguard in place. This study examined the effect of mouthguard protection in an in vitro model. A total of 97 sheep mandibular segments with incisors at four developmental stages (early deciduous, ED, n = 37; middle deciduous, MD, n = 20; late deciduous, LD, n = 18; mixed dentition, PD, n = 22) was used. Customised pressure formed mouthguards (MG) provided protection from trauma produced by a servohydraulic materials testing machine to test incisors. Injuries were examined clinically, radiographically and by dissection. Mean forces required to produce dentoalveolar injury were significantly greater in test (with MG) teeth than control (no MG) teeth. Mean forces to produce injury in test teeth decreased with resorbing root lengths. Deciduous incisors differed in injury type: subluxations and horizontal root fractures predominated in test teeth; lateral luxations and horizontal root fractures in control teeth. Predominant injuries in test and control permanent incisors were enamel infractions and subluxations. The magnitude of lateral luxation measurements of individual teeth was reduced significantly by mouthguard protection in both deciduous and permanent dentitions. The mouthguard tended to increase the mobility of the teeth it encompassed and, in some instances, promoted dentoalveolar injury of adjacent teeth.