Purpose: First, to assess the safety and efficacy of using 5-fluorouracil (5-FU) to improve trabeculectomy and phacotrabeculectomy success rates, and second, to assess the efficacy of intraoperative and reduced postoperative 5-FU following trabeculectomy compared with a more intensive course of postoperative 5-FU alone. Methods: In a retrospective, unmatched, non-randomized consecutive series study, 186 eyes of 186 patients who had filtration surgery were followed for 2 years in four groups: 51 patients had undergone trabeculectomy surgery with postoperative 5-FU, 51 had phacotrabeculectomy with postoperative 5-FU, 56 had trabeculectomy with both intraoperative and postoperative 5-FU, and 28 patients had trabeculectomy without antifibrotics. Results: At all times mean intraocular pressure (IOP) was reduced in all groups (P < 0.001 for each group). Success was defined as IOP < 16 mmHg and > 30% IOP reduction at the 2-year follow up. It was achieved in 71% of the trabeculectomy patients with intraoperative and postoperative 5-FU, 76% of the trabeculectomy group with only postoperative 5-FU, 55% of the phacotrabeculectomy/ 5-FU group, and in 29% of the trabeculectomy-only eyes (between-group differences P < 0.01). Success rates were not significantly different for the intraoperative and postoperative 5-FU trabeculectomy versus the postoperative 5-FU-only eyes, but the former had fewer postoperative 5-FU injections and corneal ulceration (P < 0.01 for both). Conclusions: 5-Fluorouracil was safe and improved trabeculectomy survival. Intraoperative 5-FU allowed fewer postoperative 5-FU injections and fewer side-effects without compromising success rates. Phacotrabeculectomy with postoperative 5-FU had a lower surgical success rate than did trabeculectomy with 5-FU and this was not statistically different from trabeculectomy without 5-FU.