Objective: To evaluate electromechanical excitation as an alternative excitation mode for middle ear transfer function (METF) measurements as well as real-time feedback in prosthetic ossicular reconstruction. Method: In eight human cadaveric temporal bones, the ossicular chain was excited using acoustic and mechanical (floating mass transducer, FMT) stimulation to determine the METF. After disconnecting the ossicular chain and reconstruction with partial or total prosthesis the METFs were measured again. Continuous FMT stimulation was then applied to improve the prosthesis’ position using real-time feedback of the METF. Results: Mechanical stimulation of ossicular vibration showed characteristic differences to acoustic excitation resulting from the force characteristics of the FMT. Furthermore, the interspecimen METF variability was greater with electromechanical than acoustic stimulation because of interspecimen variability in the FMT coupling conditions. When the METF with FMT excitation was used as a real-time feedback tool, a measurable improvement in the quality of ossicular reconstruction could be achieved. Conclusions: Mechanical excitation is an effective and suitable alternative stimulation method in experimental METF measurements. The system provides real-time feedback for ossicular reconstruction in the experimental setting. Some influencing factors still need to be distinguished for reliable measurements. However, the method does not yet meet the requirements for clinical application as an intraoperative, real-time monitoring tool. However, the system could be an excellent model for high-end cadaveric temporal bone training in ossiculoplasty.