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Background/aims This retrospective comparative study evaluated the 3-year outcomes of two mitomycin C (MMC) delivery techniques in trabeculectomy for medically uncontrolled glaucoma: innovative two-stage intra-Tenon injection versus conventional sponge application, assessing efficacy and safety parameters.Methods From January 2014 to December 2017, 110 eyes (55 intra-Tenon injections, 55 sponge applications) underwent standardised trabeculectomy by a single surgeon. Comprehensive evaluation included serial intraocular pressure (IOP) measurements, glaucoma medication requirements, surgical success rates (complete: IOP 5–21 mm Hg with ≥20% reduction without medications; qualified: with medications), needling interventions and complication profiles.Results At the 3-year follow-ups, the intra-Tenon injection group demonstrated superior outcomes: significantly greater mean IOP reduction (from 31.51 to 12.8 mm Hg and from 29.02 to 15.02 mm Hg, respectively, p=0.012), more substantial antiglaucoma medication reduction (from 4.80 to 0.85 and from 4.59 to 1.53, respectively, p=0.011), higher overall success rates (94% vs 82.22%) and lower needling frequency (1.62 vs 4.07 procedures/eye, p=0.006) than the sponge group after trabeculectomy. Complication rates were comparable, with no significant difference in visual field preservation.Conclusion The two-stage intra-Tenon MMC injection provides significantly better IOP control, reduced dependence on medications and more durable surgical outcomes than sponge application, with comparable safety, supporting its potential as the preferred method of MMC delivery in modern trabeculectomy.