The aim of this study was to evaluate the expression of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) in prostate cancer in the main tumor mass and tumor cells at the positive margin as well as the influence of these biomarkers on the biochemical recurrence of the disease in prostatectomy patients. Tissue microarrays of 120 archival prostate carcinoma samples were immunohistochemically evaluated for MMP-2 and MMP-9 expression and compared with clinicopathological parameters. Tumors with positive surgical margins showed significantly higher overall expression of MMP-9 versus tumors with negative resection margins (P = 0.0121). MMP-9 expression was significantly elevated in tumors from patients who had biochemical recurrence (P = 0.0207). In the group of patients with negative margins, MMP-9 expression above the cut-off value was significantly associated with recurrence (P = 0.0065). Multivariate analysis indicated that MMP-9 is a good predictor of biochemical recurrence (odds ratio = 10.29; P = 0.0052). Expression of MMP-2 in tumor cells was significantly higher at the positive margins than in the main tumor mass (P = 0.0301). The present results highlight the potential value of MMP-2 and MMP-9 expression for predicting the behavior of prostate tumors after prostatectomy with both positive and negative surgical margins.