Purpose: To determine the clinical and radiographic efficacy of hyaluronic acid-based cell-free scaffold applied in combination with microfracture versus microfracture alone in patients with focal osteochondral lesion of the knee joint. Methods: Clinical data of 43 patients between 24 and 55 years of age were evaluated. Hyaluronic acid-based cell-free scaffold was applied in combination with microfracture for 19 knees (group 1), whereas microfracture alone was the surgical intervention for 24 knees (group 2). All lesions were Outerbridge grade III or IV with a mean size of 3.6 +/- 1.3 cm(2). The mean follow-up time was 25.7 months. Visual analog scale (VAS), Lysholm knee score, and Tegner activity scale were the instruments used to evaluate the clinical status. Magnetic resonance observation of cartilage repair tissue (MOCART) system was used to analyze the characteristics of repair tissue. Results: Better VAS and Lysholm scores were detected in group 1 at 12 and 24 months (P=.019 and P=.025). According to the Tegner activity scale, group 1 had also better activity level at the end of 24 months after surgery (P=.020). The mean time from surgery to return to nonimpact sports activities was 7.8 months in group 1, whereas it was 9.2 months in group 2 (P=.013). Complete repair with the filling of the defect was achieved in 7 (36.8%) of the knees in group 1, whereas it was 4 (16.6%) of the knees in group 2 according to the MOCART system at 24 months. Conclusions: Single-stage regenerative cartilage surgery using hyaluronic acid-based cell-free scaffold in combination with microfracture for focal osteochondral lesions of the knee revealed promising clinical outcomes at 24 months of follow-up, but the clinical significance of the differences seen is simply not known. Level of Evidence: Level III, retrospective comparative study.