In this paper, an experimental study was conducted to investigate the effect of varying prestressing force on the nonlinearity of concrete. Prestressed concrete beams, which have been rarely studied in terms of ultrasonic nonlinearity, were prepared. When the prestressing force changes, the stress condition of the concrete inside the beam is affected. This leads to the generation of micro-cracks and a change of the mechanical properties, and as such it is necessary to introduce a nonlinear ultrasonic technique that sensitively reflects microstructural change. In the experiment, a repetitive prestressing load history, which includes maximum levels of 45%, 60%, and 75% based on the compressive strength, was designed to evaluate the effects of repetitive loading and different levels of loading on the nonlinearity. A comparison with the measured results of the linear ultrasonic method was also carried out to verify the stress dependent change of nonlinearity induced by external loads. With the experimental results, the adopted technique is found to be more sensitive to damage then conventionally used ultrasonic method. As the investigation of the methodology is effective in prestressed concrete structures, the possibility of ultrasonic nonlinearity as an indicator of prestressing force is evaluated.