For the static deflection measurement of a bridge, which is carried out for bridge rating and load-carrying capacity evaluation, sensor inaccessibility due to the fixed reference point can cause significant inconvenience and can increase installation cost and time. Therefore, an alternative indirect static deflection estimation method is proposed utilizing finite element (FE) model updating with measured acceleration data obtained from an ambient vibration test. For fast and stable optimization during model updating, a two-step model updating approach is additionally proposed. To investigate the effects of used modal quantities and their weighting factors, several cases of extensive model updating were carried out by applying several types of data sets: using only natural frequencies, using only mode shapes, and using both of them. By simulating the load test on each of the updated models, deflections were estimated and compared with the real measured deflection data in the load test. It is concluded that the static deflection can be reasonably estimated using updated FE model which is obtained by using the measured ambient acceleration data.