Antifibrotic agents are used during trabeculectomy (TRAB) to improve long-term surgical success by reducing fibrosis of subconjunctival and episcleral tissue. The purpose of this meta-analysis was to directly compare the efficacy and safety of the two most frequently used antifibrotic agents, Mitomycin C (MMC) vs. 5-FU, when used as adjunctive therapy for TRAB. A systematic review was performed in April 2011. All randomised controlled trials (RCTs) comparing 5-FU directly with MMC were eligible for inclusion. Other inclusion criteria included age ≥18 years, glaucomatous optic disc features and intraocular pressure (IOP) ≥21. Five RCTs met the inclusion criteria, totalling 416 participants. MMC was associated with a statistically significant lower mean IOP level following TRAB than 5-FU (-2.17mmHg; 95%CI -3.26 to -1.08, P<0.001). Subgroup analysis revealed that a significant efficacy benefit was observed with high dose adjuvant MMC (>0.2mg/ml) compared with adjuvant 5-FU but no significant difference was observed between low dose adjuvant MMC (≤0.2mg/ml) compared with adjuvant 5-FU. There was no significant difference in the occurrence of wound leak, persistent hypotony, endophthalmitis, choroidal effusion and decrease in visual acuity of more than two lines between MMC and 5-FU. The MMC group experienced a significantly lower incidence of postoperative corneal epithelial defects than the 5-FU group (4/111 [3.6%] vs. 13/109 [12%] participants; P=0.021). The authors conclude TRAB with MMC is associated with higher rates of surgical success and is not associated with increased postoperative complications compared with TRAB with 5-FU.