The aim of this study was to investigate the association of serum hyaluronidase and nitric oxide (NO) levels with arterial stiffness in patients with hypertension (HT) and diabetes mellitus (DM). A total of 101 patients with diagnosis of DM and HT were enrolled in this study. The patients were divided into three groups as follows: only hypertensive (I), only diabetic (II) and both diabetic and hypertensive (III). Serum hyaluronidase levels were negatively correlated with aortic strain (AS) and aortic distensibility (AOD) in all groups, whereas a significant positive correlation was noted between serum hyaluronidase levels and aortic strain index (ASI) (all p-values < 0.05). There was a significant negative correlation between serum hyaluronidase and NO levels in all patients (p < 0.001). When the correlation between serum hyaluronidase and serum NO levels was investigated in the individual patient groups, a negative correlation was found in groups I, II and III (p = 0.017, p < 0.001 and p < 0.001, respectively). A significant relationship between plasma hyaluronidase level and parameters of aortic stiffness was found in patients with HT and/or DM. We suggest that the pathophysiological mechanisms responsible for the development of arterial stiffness in subjects with impaired endothelial function may involve pathological changes in the HA metabolism.