The purpose of our study was to assess the clinical and imaging outcome of autologous matrix-induced chondrogenesis (AMIC) technique consisting of microfractures followed by the filling of osteochondral lesions of the talus (OLTs) with a cell-free biphasic collagen-hydroxyapatite osteochondral scaffold (MaioRegen). Sixteen patients (eight males, age: 42.6 ± 18.4, range 14–74) with OLT repaired using AMIC technique, with implantation of MaioRegen, were clinically evaluated through the American Orthopedic Foot and Ankle Society Score (AOFAS) and a 10-point Visual Analogue Scale (VAS) pain score after a mean follow-up of 30 ± 16.9 months. The MRI examinations were performed 12 and 24 months after surgery. A paired t-test was applied to compare pre- and post-operative clinical findings (VAS and AOFAS) and Magnetic resonance observation of cartilage repair tissue (MOCART) score changes in the follow-up. To assess the correlation between variation of AOFAS and MOCART scores, the Pearson’s correlation coefficient was calculated. No complications after surgery were encountered. From pre-operative to post-operative values, there was a significant (P < 0.001) reduction of mean VAS pain score (6.3 ± 0.9,range: 4–8 and 2.9 ± 1.8,range: 0–6, respectively) and increase of AOFAS score (60.2 ± 7.8,range: 50–74 and 77.4 ± 16.2,range: 50–100, respectively). Among 16 patients, six (37%) were not satisfied at the end of follow-up, six (37%) were moderately satisfied and four (25%) were highly satisfied. The treatment was considered failed in five out of 16 patients (31%). Among them, four (25%) required re-interventions with implantation of ankle prostheses, whereas one patient was treated with a further AMIC technique combined with autologous bone graft and platelet-rich plasma. The mean MOCART score was 41.9 ± 14.6 (25–70) 12 months after surgery and 51.9 ± 11.6 (30–70) after 24 months, with a statistically significant increase (P = 0.012). However, no correlation was seen between AOFAS and MOCART changes (r = 0.215, p = 0.609). The high rates of treatment failure encountered in our study using MaioRegen need to be confirmed by larger studies and should induce the scientific community questioning the reliability of this biomimetic scaffold for the treatment of OLTs.