<b><i>Introduction:</i></b> The effect of the continuous forced expiration action of players of wind instruments to produce sound, on the eustachian tube functions and the middle-ear resonance frequency (RF), has not been investigated in the literature to date. The aim of this study is to evaluate eustachian tube functions and the middle-ear RF of players of wind instruments. <b><i>Methods:</i></b> In this prospective case-control clinical study, a study group of 28 players of wind instruments in the orchestra (28 participants, 56 ears) and a control group of 34 volunteers (34 participants, 68 ears) were included. The eustachian function of wind instrument players in a symphony orchestra was measured using an automatic eustachian tube function test in acoustic tympanometry and the RF of the middle ear was determined in multifrequency tympanometry. <b><i>Results:</i></b> There was a statistically significant difference among the musicians, especially in players of woodwind instruments, in terms of dysfunction of the eustachian tubes (<i>p</i> = 0.048). In the musicians, the pre- and postperformance RF mean values for all ears were 925 and 1,020 Hz, respectively, and these were significantly different (<i>p</i> = 0.004). <b><i>Conclusion:</i></b> This is the first study to uses multifrequency tympanometry to examine the middle-ear RF and eustachian tube function of wind instrument musicians in an orchestra. Eustachian tube dysfunction was found to be more prominent and a higher RF of the middle ear was seen after a performance, especially in players of wood wind instruments. However, the effect of these on the professional performance of players of wind instruments should be investigated in future work.