The development of techniques able to characterize and map the pressure field is crucial for the widespread use of acoustofluidic devices in biotechnology and lab-on-a-chip platforms. In fact, acoustofluidic devices are powerful tools for driving precise manipulation of microparticles and cells in microfluidics in non-contact modality. Here, we report a full and accurate characterization of the movement of particles subjected to acoustophoresis in a microfluidic environment by holographic imaging. The particle displacement along the direction of the ultrasound wave propagation, coinciding with the optical axis, is observed and investigated. Two resonance frequencies are explored, varying for each the amplitude of the applied signal. The trajectories of individual tracers, accomplished by holographic measurements, are fitted with the theoretical model thus allowing the retrieval of the acoustic energy densities and pressure amplitudes through full holographic analysis. The absence of prior calibration, being independent of the object shape and the possibility of implementing automatic analysis make the use of holography very appealing for applications in devices for biotechnologies.