. The study examines an Er:YAG laser (2940 nm) and different application systems of the CO2 laser (10 600 nm) with regard to their suitability for a one-shot laser myringotomy of an adequate perforation size (∼2 mm). The laser–tissue interaction of the Er:YAG laser and the CO2 laser in fresh tympanic membranes of horses (thickness: 80–100 µm) as well as in formalin-fixed human tympanic membranes (thickness: 100 µm) is studied correlating perforation diameters to the applied power/energy density and the effects demonstrated by light and scanning electron microscopy are analysed. Using the Er:YAG laser with a focused laser beam (spot diameter: 400 µm) or with a maximally defocused laser beam (spot diameter: 1600 µm) perforations of an adequte size (2 mm) can only be achieved with multiple laser pulses. Histological studies disclose only minimal thermic side effects in the adjacent tissue in both specimens. If the CO2 laser radiation is transmitted via a silver halide polycrystalline fibre (diameter: 900 µm) a maximal perforation diameter of 1300 µm is achieved with significant thermic side effects such as coagulation. Using an Acuspot™ 710 micromanipulator (focused beam diameter: 180 µm) combined with a SilkTouch™ scanner a maximal perforation diameters of 1700 µm can be achieved in horse tympanic membrane with one laser pulse. A prototype of a hand-held CO2 laser otoscope in combination with the SilkTouch™ scanner is suitable for performing laser myringotomies with a diameter of 2 mm with a single laser pulse in fresh horse tympanic membrane.