Excessive noise in the work environment has been associated with extra-auditory symptoms, which can have harmful long-term effects on individuals. The purpose of this study was to identify noise levels in neonatal intensive care units and investigate their impact on the occurrence of stress among healthcare professionals, using cortisol levels as a biomarker for Burnout Syndrome. This descriptive, observational, and cross-sectional study was conducted in four public teaching hospitals in Fortaleza, Ceará, Brazil. Sound pressure levels in the environment were measured, and questionnaires were administered to collect sociodemographic data and assess perceptions of the work environment and Burnout symptoms. Saliva samples were collected at the beginning and end of work shifts for cortisol quantification. The average sound pressure ranged from 59.9 to 66.4 dB(A), exceeding the recommended levels set by Brazilian and international legislation. Among the 256 participants, the average age was 39.4 years, with 95% being female. The majority (70.9%) were nurses, and 22.7% were physicians. There was no significant association found between noise and Burnout Syndrome, nor with changes in cortisol levels. However, a significant association was observed between the perception of excessive noise and the sensation of a stressful work shift (p = 0.012). All evaluated professionals displayed symptoms of Burnout. The high sound pressure levels indicated that the assessed environments did not meet the recommended standards for acoustic comfort, and this was associated with the participants’ perception of stressful work shifts. While Burnout symptoms were evident in our participants, it was not possible to confirm a correlation with high noise levels.