Prestress force (PF) and the applied loads are two significant aspects that relate to the safety of in-service prestressed concrete bridges (PCBs). The PF directly governs the load carrying capacity of the bridge, and the prestress losses would decrease the effective value of PF which would in turn reduce the strength of the bearing members or even lead to bridge failure; while the applied loads caused by vehicles are critical for bridge diagnosis and maintenance. Overloading would damage the bridge deck and substructure resulting in a large reduction of the structure’s lifespan. These potential safety hazards raise the need to monitor these two forces and maintain them in a safety range. In this paper, the PF and unknown load applied on a simply supported box girder bridge are determined simultaneously through a synergic identification method which takes advantage of virtual distortion method (VDM), Duhamel integral and load shape function (LSF). The feasibility of this method is examined in a comprehensive laboratory test. Results show that the method attains a good degree of accuracy and efficiency in assessing PF and stationary force, and has good robustness to noise.