Driving behavior in a highway tunnel could be affected by external environmental factors like light, traffic flow, and acoustic environments, significantly when these factors suddenly change at the moment before and after entering a tunnel. It will cause tremendous physiological pressure on drivers because of the reduction of information and the narrow environment. The risks in driving behavior will increase, making drivers more vulnerable than driving on the regular highways. This research focuses on the usually neglected acoustic environment and its effect on drivers' physiological state and driving behavior. Based on the SIMLAB driving simulation platform of a highway tunnel, 45 drivers participated in the experiment. Five different sound scenarios were tested: original highway tunnel sound and a mix of it with four other sounds (slow music, fast music, voice prompt, and siren, respectively). The subjects' physiological state and driving behavior data were collected through heart rate variability (HRV) and electroencephalography (EEG). Also, vehicle operational data, including vehicle speed, steering wheel angle, brake pedal depth, and accelerator pedal depth, were collected. The results indicated that different sound scenarios in the highway tunnel showed significant differences in vehicle speed (p = 0.000, η2 = 0.167) and steering wheel angle (p = 0.007, η2 = 0.126). At the same time, they had no significant difference in HRV and EEG indicators. According to the results, slow music was the best kind of sound related to driving comfort, while the siren sound produced the strongest driver reaction in terms of mental alertness and stress level. The voice-prompt sound most likely caused driver fatigue and overload, but it was the most effective sound affecting safety. The subjective opinion of the drivers indicated that the best sound scenario for the overall experience was slow music (63%), followed by fast music (21%), original highway tunnel sound environment (13%), and voice-prompt sound (3%). The findings of this study will be valuable in improving acoustic environment quality and driving safety in highway tunnels.