Abstract <p>Under conditions of ground-based simulation of the physiological effects of microgravity (7-day dry immersion), an experimental study was conducted to assess the functional state of the auditory system and the composition of ENT microflora in ten healthy male volunteers aged 25–36 years. The functional state of the auditory system was evaluated using distortion-product otoacoustic emission (DPOAE) recordings and tympanometry. The following parameters were assessed: signal-to-noise ratio (SNR) of the otoacoustic response (dB), middle ear pressure (daPa), and maximum tympanometric compliance (mL). To assess the individual state of the ENT microflora, samples were collected from the mucosal membranes of the nasal cavity and pharynx, as well as from the skin of the external auditory canals. Microbial cultures were identified and quantitatively analyzed. Changes in microbial composition were assessed using the eubiotic index. Alterations in the functional state of the auditory system in the examined volunteers were characterized by a statistically significant (<i>p</i> ≤ 0.05) decrease in the DPOAE signal-to-noise ratio (SNR) in the low-frequency range, a statistically significant (<i>p</i> ≤ 0.05) increase in middle ear pressure, and a clear trend toward a decrease in maximum tympanometric compliance. According to the dynamic assessment of ENT microbiocenosis, adverse changes were observed, characterized by a reduction in commensal microorganisms and an increase in opportunistic microorganisms. The eubiotic index values were below 1. These findings indicate a potential adverse effect of simulated microgravity on both the functional state and microbial composition of the human ENT system. The use of these methods for dynamic assessment and monitoring of the auditory system and ENT microbiocenosis in cosmonauts during actual spaceflight is considered justified.</p>