The Neonatal Intensive Care Unit (NICU) could be a risk factor for the development of preterm neonates due to the stressful procedures they undergo. Stress-related behaviors must be managed through environmental strategies that support regulating the neonates' biobehavioural system to minimize the negative impact on their development. The study aimed to compare the NICU environment's stressful procedures and developmental care strategies and the stress and self-regulation behaviors of preterm neonates in groups differentiated by the NICU environmental design. The sample comprised 20 preterm neonates hospitalized in a NICU with an open-bay model (OB NICU) and 20 preterm neonates hospitalized in a single-family room model (SFR NICU). The stressful procedures were assessed by the Neonatal Infant Stressor Scale (NISS). The developmental care strategies and the preterm neonates' stress and self-regulation behaviors were assessed using a structured observational protocol. The between-group comparison was performed by the Mann-Whitney test, and the significance level was set at 5%. Both NICUs had similar stressful procedures and developmental care approaches. However, the preterm neonates hospitalized in the SFR NICU exhibited significantly fewer total stress behaviors, and specifically in the motor system, compared to those in the OB NICU. Additionally, the preterm neonates hospitalized in the SFR NICU exhibited significantly more total self-regulation behaviors, and specifically in the behavioral state system, compared to those in the OB NICU. The findings showed that the single-family room NICU model was consistent with the environmental protection of biobehavioural regulation in preterm neonates hospitalized in the NICU.