| Summary: | (1) Background: Mitomycin C (MMC) is commonly used during trabeculectomy. However, there is no consensus on which concentration should be used. We aimed to compare the efficacy and safety of 0.2 mg/mL and 0.4 mg/mL of MMC in eyes undergoing trabeculectomy. (2) Methods: Thirty-six eyes (36 glaucoma patients) were randomized to undergo a trabeculectomy with 0.2 mg/mL or 0.4 mg/mL of MMC. The success rate was evaluated according to three criteria: (A) intraocular pressure (IOP) ≤18 mmHg and IOP reduction ≥20%; (B) IOP ≤15 mmHg and IOP reduction ≥25%; (C) IOP ≤12 mmHg and IOP reduction ≥30%. Cox’s proportional hazard model was used to identify the predictive factors for failure. Immunohistochemical procedures for matrix metalloproteinase (MMP) were performed on Tenon’s tissue. Bleb morphology was evaluated. Safety was assessed based on the incidence of complications. (3) Results: Of the 36 eyes, 19 underwent trabeculectomy with 0.2 mg/mL of MMC and 17 with 0.4 mg/mL. The success rates were 75%, 67%, and 47% at 6 months for criteria A, B, and C, respectively. There were no significant differences between the two groups. High MMP-9 staining and low preoperative IOP were associated with failure (hazard ratio (HR), 5.556; <i>p</i> = 0.033, and HR, 0.936; <i>p</i> = 0.033). Complications included hypotony in two eyes (6%), hyphema in one eye (3%), and choroidal detachment in one eye (3%). (4) Conclusions: Trabeculectomy with 0.2 mg/mL and 0.4 mg/mL of MMC showed similar IOP-control effects to those recorded in previous studies, along with a low rate of complications. There was no significant difference in efficacy or safety between the 0.2 mg/mL and 0.4 mg/mL MMC groups.
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