Purpose Accidental injury to the parathyroid glands (PTGs) is common during thyroid and parathyroid surgery. To overcome the limitation of naked eye in identifying the PTGs, intraoperative autofluorescence imaging has been embraced by an increasing number of surgeons. The aim of our study was to describe the technique and assess its utility in clinical practice. Methods Near-infrared (NIR) autofluorescence imaging was carried out during open parathyroid and thyroid surgery in 25 patients (NIR group), while other 26 patients underwent traditional PTG detection based on naked eye alone (NO-NIR group). Primary variables assessed for correlation between traditional approach and autofluorescence were number of PTGs identified and incidence of postoperative hypoparathyroidism (hypoPT). Results 81.9% of PTGs were detected by means of fluorescence imaging and 74.5% with visual inspection alone, with an average of 2.72 PTGs visualized per patient using NIR imaging versus approximately 2.4 per patient using naked eye ( p  = 0.38). Considering only the more complex total thyroidectomies (TTs), the difference was almost statistically significant ( p  = 0.06). Although not statistically significant, the observed postoperative hypoPT rate was lower in the NIR group. Conclusion Despite the limitations and technical aspects still to be investigated, fluorescence seems to reduce this complication rate by improving the intraoperative detection of the PTGs.