Matrix degradation and remodeling occurs during wound healing, thereby aiding tissue repair, angiogenesis, and cell migration. It is dependent on the balance between proteinases and their inhibitors, namely the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Acute wound fluid samples (n = 58 patients) were collected daily from the intraperitoneal drain placed after colorectal surgery from the first postoperative day until drain removal. Three laboratory techniques were performed: enzyme linked immunosorbent assays (MMP-1, MMP-3, TIMP-1, TIMP-2), gelatinase activity assays (MMP-2, MMP-9), and quenched fluorescent substrate hydrolysis (total MMP activity). Levels were correlated with each postoperative day, wound healing, and surgical outcome (p < 0.05, Spearman's correlation). Significant negative (MMP-9, MMP-3, MMP-8, TIMP-2, total MMP activity) and positive (MMP-2, TIMP-1) correlations were observed with the postoperative day, e.g., total MMP-9: day 1, median, 121 (range, 12-189) ng/ml; day 3, 46 (8-179); day 5, 31 (0-155), day 7, 20 (6-58). Differences were also observed with the type of operation, estimated blood loss, and length of operation and with postoperative complications. MMPs and TIMPs are involved in wound healing after elective colorectal cancer surgery and their levels in drain fluid may act as markers of wound healing and surgical outcome.