We retrospectively evaluated a consecutive series of 45 patients (45 eyes) who underwent trabeculectomy augmented with a single intra-operative 5 minute application of 5-fluorouracil (5-FU; 25 mg/ml). All patients were at an increased risk of subconjunctival fibrosis and surgical failure. The mean follow-up period was 24 months (range 12-42, SD 6.9). The mean pre-operative intraocular pressure (IOP) was 29.1 mmHg (SD 6.1) and the mean IOP at the last post-operative visit was 16.6 mmHg (SD 6.4) (p < 0.0001) with a mean IOP reduction of 42%. The number of medications reduced from a mean of 2.3 (SD 0.7) pre-operatively, to 0.8 (SD 0.7) post-operatively (p < 0.0001) and 22 eyes (49%) required no topical treatment for IOP control. An IOP of 21 mmHg or less with or without medications was achieved in 80% of cases. There was no significant difference in final IOP or success rate over time between low- and high-risk patients, although the low-risk patients did better in the first 12-18 months. Complications included hypotony maculopathy in 2 cases (4%), leaking bleb in 5 cases (11%) and giant bleb in 1 case (2%), giving a total of 8 cases (18%) with bleb-related sequelae. In the short to medium term, a single per-operative application of 5-FU is a useful adjunctive treatment during glaucoma filtering surgery for low- to moderate-risk cases, although a steady increase in the failure rate was associated with increasing length of follow-up.