The purpose of this study was to determine whether delayed, postoperative, intraperitoneal treatment with 5-fluorouracil (5-FU) plus interferon-alpha-2a (IFN) has adverse effects on colonic healing, as does early treatment. Seventy male Wistar rats underwent colonic anastomoses. The rats were randomized to one of four groups. Early intraperitoneal injection was given to groups 1 and 2 which was repeated once daily for the first 3 postoperative days. Treatment was delayed in groups 3 and 4, from the 4th to the 7th postoperative day. A 0.9% NaCl solution was injected in the rats of control groups 1 and 3. In groups 2 and 4, we infused 5-FU (20 mg/kg/day) and IFN (45,000 IU/kg/day). All the animals were sacrificed on the 8th postoperative day. The anastomotic rupture rate was significantly higher in the rats of group 2 compared to control group 1 (p < 0.05), while there were no differences between groups 3 and 4 (p > 0.05). Abscess formation and adhesions were more frequent in group 2 compared to control group 1, while no differences were observed between groups 3 and 4. Anastomotic bursting pressure was statistically significantly lower in the rats of group 2 compared to group 1 (p < 0.05); no differences were noticed between groups 3 and 4 (p > 0.05). Simultaneous histologic evaluation showed a more profound inflammatory reaction and delayed anastomotic healing in group 2 compared to control group 1; there were, however, no differences between groups 3 and 4. In conclusion, the immediate, postoperative, intraperitoneal injection of 5-FU plus IFN impairs colonic healing while delayed treatment (starting on the 4th postoperative day) has no adverse effects on wound healing.