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Abstract Purpose The treatment of osteochondral lesions is challenging and no consensus has been established about the best option for restoring both cartilage and subchondral bone. Multilayer collagen‐hydroxyapatite scaffolds have shown promising clinical results, but the outcome at a follow‐up longer than 5 years still has to be proved. The aim was to evaluate the clinical outcome of patients with a knee isolated osteochondral lesion treated with a biomimetic three‐layered scaffold at a minimum 5 years of follow‐up. Methods Twenty‐nine patients (23 males and 6 females, mean age 31.5 ± 11.4 years) were evaluated retrospectively before surgery, at 1 and 2 years and at last follow‐up (FU). Visual Analog Scale (VAS) for pain, International Knee Documentation Committee (IKDC) Subjective Score, Tegner‐Lysholm Knee Scoring Scale and Tegner Activity Level Scale were collected. Mean FU was 7.8 ± 2.0 years (min 5.1 ‐ max 11.3). The etiology of the defect was Osteochondritis Dissecans or osteonecrosis (17 vs 12 cases). Results At 12 months FU the IKDC score improved from 51.1 ± 21.7 to 80.1 ± 17.9 (p < 0.01), Tegner Lysholm Score from 59.9 ± 17.3 to 92.5 ± 9.0 (p < 0.01), VAS from 6.1 ± 2.1 to 1.7 ± 2.3 (p < 0.01) and Tegner Activity Level Scale from 1.6 ± 0.5 to 4.9 ± 1.7 (p < 0.01). The results remained stable at 24 months, while at last FU a statistically significant decrease in IKDC, Tegner Lysholm and Tegner Activity Scale was recorded, though not clinically relevant. Patients under 35 achieved statistically better outcomes. Conclusions The use of a cell‐free collagen‐hydroxyapatite osteochondral scaffold provides substantial clinical benefits in the treatment of knee osteochondral lesions at a minimum follow‐up of 5 years, especially in patients younger than 35 years. Level of evidence Level IV.