In this paper, it is aimed to determine the seismic behaviour of highway bridges by nondestructive testing using ambient vibration measurements. Eynel Highway Bridge which has arch type structural system with a total length of 216 m and located in the Ayvacik county of Samsun, Turkey is selected as an application. The bridge connects the villages which are separated with Suat Ugurlu Dam Lake. A three dimensional finite element model is first established for a highway bridge using project drawings and an analytical modal analysis is then performed to generate natural frequencies and mode shapes in the three orthogonal directions. The ambient vibration measurements are carried out on the bridge deck under natural excitation such as traffic, human walking and wind loads using Operational Modal Analysis. Sensitive seismic accelerometers are used to collect signals obtained from the experimental tests. To obtain experimental dynamic characteristics, two output-only system identification techniques are employed namely, Enhanced Frequency Domain Decomposition technique in the frequency domain and Stochastic Subspace Identification technique in time domain. Analytical and experimental dynamic characteristic are compared with each other and finite element model of the bridge is updated by changing of boundary conditions to reduce the differences between the results. It is demonstrated that the ambient vibration measurements are enough to identify the most significant modes of highway bridges. After finite element model updating, maximum differences between the natural frequencies are reduced averagely from 23% to 3%. The updated finite element model reflects the dynamic characteristics of the bridge better, and it can be used to predict the dynamic response under complex external forces. It is also helpful for further damage identification and health condition monitoring. Analytical model of the bridge before and after model updating is analyzed using 1992 Erzincan earthquake record to determine the seismic behaviour. It can be seen from the analysis results that displacements increase by the height of bridge columns and along to middle point of the deck and main arches. Bending moments have an increasing trend along to first and last 50 m and have a decreasing trend long to the middle of the main arches.