Purpose: To describe the experience of our centre (Careggi University Hospital, Florence, Italy) in using a heads-up three-dimensional (3D) surgical viewing system in vitreoretinal surgery, making a comparison with the conventional microscope surgery. Methods: We retrospectively analyzed data taken from 240 patients (240 eyes) with surgical macular diseases (macular hole and epiretinal membrane), retinal detachment or vitreous hemorrhage who underwent vitreoretinal surgeries, by means of the NGENUITY 3D Visualization System (Alcon Laboratories Inc., Fort Worth, TX, USA), in comparison with 210 patients (210 eyes) who underwent vitreoretinal surgeries performed using a conventional microscope. All surgeries were performed with standardized procedures by the same surgeons. We analyzed data over a follow-up period of 6 months, comparing the surgical outcomes (best-corrected visual acuity, anatomical success rate and postoperative complication rate) between the two groups. Results: the 3D group included 74 patients with retinal detachment, 78 with epiretinal membrane, 64 with macular hole and 24 with vitreous hemorrhage. There were no significant differences in the demographic and clinical characteristics between the 3D group and the conventional group. We found no significant differences in outcome measures at three and six months follow-up between the two groups (p-value ≥ 0.05 for all comparisons). Surgery durations were similar between the two groups. Conclusions: In our experience, a heads-up 3D surgical viewing system provided comparable functional and anatomical outcomes in comparison with conventional microscope surgery, proving to be a valuable tool for vitreoretinal surgery in the treatment of different retinal diseases.