Abstract The effect of subconjunctival injection of 5‐fluorouracil (5‐FU) after trabeculectomy was studied retrospectively in 205 eyes of 168 patients. A life table analysis of the surgical outcome was based on the type of glaucoma and age related differences, and a comparison was made with patients who had trabeculectomy without subconjunctival 5‐FU. The success rate at 30 months after trabeculectomy with 5‐FU therapy was considerably higher in primary open‐angle glaucoma at 93.6% (72.7%), secondary glaucoma at 88.9% (72.4%), and refractory glaucoma at 72.2% (32.5%) with (or without) the use of ocular hypotensive drops when compared with historical control groups treated without 5‐FU (60.0% (41.7%), 35.5% (24.0%), and 18.0% (8.0%), respectively). In patients aged over 70 years, no statistically significant improvement could be demonstrated with the use of 5‐FU after trabeculectomy in primary open‐angle glaucoma. Our study may provide data on the appropriate dosage and indications for the use of this drug after glaucoma surgery.