Background Mitomycin C (MMC), as a wound-healing modulatory factor, can be applied during trabeculectomy to increase its success rate. This may affect the morphology and function of corneal endothelium as well as its central cell count.Aim The aim of this study was to investigate the effect of intracameral injection of viscoelastic combined with subconjunctival application of viscoelastic in trabeculectomy with MMC performed for uncontrolled chronic primary angle-closure glaucoma (PACG) in order to decrease the MMC side effects on the corneal endothelial cells.Patients and methods This is a prospective randomized comparative case-series study that included 70 eyes of 70 patients with uncontrolled chronic PACG. The patients were referred for glaucoma surgery in a tertiary hospital. They were randomized into two groups with 35 eyes in each group: group 1 included 35 eyes that underwent subscleral trabeculectomy with MMC with intracameral injection of dispersive viscoelastic and subconjunctival application of cohesive viscoelastic, while group 2 included 35 eyes that underwent subscleral trabeculectomy with MMC application only. In both groups, endothelial cells were assessed by specular microscopy preoperatively and postoperatively. The outcome measures included the corneal endothelial cell density, hexagonality (HEX), coefficient of variation of cell size, central corneal thickness, as well as intraocular pressure (IOP).Results The mean age of the patients was 53.43±5.14 years in group 1 and 52.54±4.2 years in group 2. The IOP decreased from 31.09±3.17 mmHg preoperatively to 11.2±0.8, 11.8±0.9, 11.9±0.9, and 11.9±0.9 mmHg at 1, 3, 6, and 12 months postoperatively, respectively, in group 1, while it decreased from 30.5±3.7 mmHg preoperatively to 13.7±1.1, 14.4±0.9, 14.6±0.8, and 14.7±0.7 mmHg at 1, 3, 6, and 12 months postoperatively, respectively, in group 2. The corneal endothelial cell density significantly decreased from 2482±179 cells/mm2 preoperatively to 2350±62 cells/mm2 at 12 months postoperatively (P<0.001) in group 1, while it significantly decreased from 2461.17±41.58 cells/mm2 preoperatively to 2273±37.5 cells/mm2 at 12 months postoperatively (P<0.001) in group 2, with a significant difference between the two groups (P<0.001). The coefficient of variation as a measurement of polymegathism was significantly increased from 27.21±0.41 preoperatively to 27.73±0.478 postoperatively in group 1 (P<0.001), while it significantly increased from 27.18±0.40 preoperatively to 27.72±0.4 postoperatively in group 2 (P<0.001). HEX, which is an indication of variability in HEX shape, was significantly decreased from 71.5±0.4% preoperatively to 62.0±1.8% at 1 month and then significantly increased to 68.2±1.8% at 12 months postoperatively in group 1 (P<0.001). On the other hand, it decreased from 71.55±0.40% preoperatively to 62.0±1.75% at 1 month and then significantly increased to 68.17±1.77% at 12 months postoperatively in group 2 (P<0.001). Intracameral combined with subconjunctival application of viscoelastic significantly reduced the IOP in group 1 to 11.94±0.91 mmHg more than in group 2 (14.69±0.76 mm Hg) at the end of the follow-up period (P<0.001).Conclusion Intracameral injection of dispersive viscoelastic, in addition to subconjunctival injection of cohesive viscoelastic, may be a good method to preserve the corneal endothelium in PACG cases, for which trabeculectomy with MMC application was performed. Moreover, this extrastep may increase the trabeculectomy success rate and reduce its complications.