Aim: Emerging evidence favors the important role of antioxidants, matrix metalloproteinases (MMPs), and nitric oxide (NO) in the healing of diabetic wounds. There is a lack of substantial evidence regarding the effects of negative pressure on antioxidants, MMPs and NO in chronic wounds associated with diabetes. Methods: A total of 55 type 2 diabetic patients with leg ulcers were divided into 2 groups: a limited access dressing (LAD) group (n = 27) and a conventional dressing group (n = 28). Levels of hydroxyproline, total protein, MMP-2 and MMP-9, NO and antioxidants including reduced glutathione (GSH) and the oxidative biomarker malondialdehyde (MDA) were measured in the granulation tissue at days 0 and 10. Changes in levels between the LAD and conventional groups were determined by the Student's t-test. Results: After 10 days of treatment, the LAD vs. conventional dressing group showed increase in the levels of hydroxyproline (mean ± standard deviation = 55.2 ± 25.1 vs. 29.2 ± 1, P < 0.05), total protein (12.8 ± 6.5 vs. 8.34 ± 3.2, P < 0.05), NO (1.13 ± 0.52 vs. 0.66 ± 0.43, P < 0.05), GSH (7.0 ± 2.4 vs. 6.6 ± 2.2, P < 0.05) and decrease in MMP-2 (0.47 ± 0.33 vs. 0.62 ± 0.30, P < 0.05), MMP-9 (0.32 ± 0.20 vs. 0.53 ± 0.39, P < 0.05) and MDA (6.8 ± 2.3 vs. 10.4 ± 3.4, P < 0.05). Conclusion: When compared to conventional dressings, LAD induces biochemical changes by significantly increasing the levels of hydroxyproline, total protein, NO and antioxidants levels, and significantly reducing MMPs (MMP-2 and MMP-9) and an oxidative biomarker in diabetic wounds. These biochemical changes are thought to favor diabetic wound healing.