The natural frequencies of composite bridges are affected by temperature variations and boundary conditions, presenting challenges for damage-detection methods based on these parameters. In this study, the individual and combined effects of temperature variations and boundary condition changes were investigated both numerically and experimentally. Temperature variations included uniform temperature fields and temperature gradients within the bridge section. For modal testing, a temperature-controllable composite girder with adjustable supports was developed. Numerical analysis further evaluated the influence of temperature and boundary condition changes on natural frequencies. The results demonstrated that changes in geometric stiffness due to the combined effects of temperature and boundary conditions, as well as the temperature-dependent behavior of the elastic modulus, significantly affected the natural frequencies. • This study is the first to experimentally investigate the combined effects of temperature and boundary conditions on frequency responses of a composite girder. • The Natural frequency of composite bridges decreases with temperature, mainly due to temperature-dependent elastic modulus. • Under temperature gradient, the H-R boundary shows a higher frequency reduction rate than H-H, influenced by greater convex deformation. • For uniform temperature, both boundary conditions have similar frequency reduction rates, primarily driven by elastic modulus changes. • Linear regression models accurately predict frequency responses to temperature and boundary conditions.