Purpose: To evaluate the long-term outcome of trabeculectomy with intra- and postoperative 5-fluorouracil (5-FU) application in glaucoma. Methods: Eighty-six patients with glaucoma planned for primary trabeculectomy with 5-FU and a minimum follow-up of 3 years were retrospectively analyzed. Success rates, postsurgical 5-FU injections, needling procedures, and complications were analyzed. Results: Mean intraocular pressure (IOP) decreased from 27.2 ± 6.7 to 13.2 ± 4.2 mm Hg at 1 year and 13.8 ± 3.7 mm Hg at the 3-year follow-up. The complete success rates (no IOP-lowering medication) were 83, 79, 73, and 45% at 1 year for IOP ≤21, ≤18, ≤16, and ≤12 mm Hg, respectively, and 64, 59, 56, and 20% for these criteria at 3 years. The average number of medications decreased from preoperatively 2.9 ± 1.4 to 0.2 ± 0.5 at 1 year and 0.7 ± 1.1 at 3 years. During the first 6 months, subconjunctival 5-FU injections were performed in 49 cases. Eleven patients underwent bleb needling during the first 6 months and 13 patients underwent the procedure between the 6th month and the 3rd year. Malignant glaucoma and bleb-related endophthalmitis occurred in 1 patient each. Conclusions: Trabeculectomy with 5-FU is an efficient surgical procedure for glaucoma treatment when combined with intensified postsurgical care.