This study aimed to evaluate corneal stromal and endothelial alterations after vitrectomy using in vivo confocal microscopy (IVCM), focusing on the implications of different surgical techniques and tamponades. In this prospective cohort study, a total of 99 eyes from 98 consecutive patients scheduled for vitrectomy were initially enrolled. Each subject underwent a comprehensive ophthalmic evaluation, including fundus examination, intraocular pressure assessment, and IVCM imaging. These evaluations were conducted preoperatively, as well as at 1, 3, 6, 9, and 12 months following surgery. Data were analyzed to correlate corneal cellular alterations with vitrectomy techniques and tamponades. Data from 80 eyes of 79 patients were used in the final analysis stratified into six groups based on the combination of surgical approach and tamponade used. Postoperatively, compared to baseline, posterior stromal cell density (SCD) significantly increased in patients who underwent phacoemulsification (Phaco) and intraocular lens (IOL) implantation with C3F8 tamponade, while anterior SCD significantly decreased in patients treated with Phaco and silicone oil (SiO2). The prevalence of keratic precipitates, endothelial nucleation, and dark spots in the corneal endothelial layer significantly increased post-surgery and did not return to the preoperative baseline by the end of the follow-up period, especially in the group treated with Phaco and C3F8 tamponade. Endothelial cell loss was significantly higher at 3 months post-surgery, with rates of 9.16% for air tamponade, 8.86% for C3F8, 2.93% for SiO2, 9.24% for Phaco and C3F8, 11.58% for Phaco with IOL and C3F8, and 11.99% for Phaco with SiO2. By the end of the 12-month follow-up, losses were 8.12% for air, 9.39% for C3F8, 16.38% for SiO2, 3.39% for Phaco and C3F8, 10.65% for Phaco with IOL and C3F8, and 14.40% for Phaco with SiO2. Our IVCM-based study offers insights into the dynamic responses of corneal cells to vitreoretinal surgeries, focusing on the effects of Air, C3F8, and SiO2 tamponades, with and without Phaco. The distinct patterns of response observed in the C3F8 and SiO2 groups, both with and without Phaco, underscore the importance of carefully choosing the surgical procedure based on individual fundus conditions and implementing tailored postoperative care based on the tamponade used. Our findings lay the groundwork for further studies on the long-term effects of tamponade materials and the development of targeted therapies to mitigate corneal reactions related to vitrectomy.