Single-surgery treatment of knee-cartilage lesions with primary autologous articular chondrocytes (PCs) and mononuclear bone-marrow cells (MNCs) is promising, but midterm clinical results are scarce. We here report KOOS and IKDC outcomes, as well as patient satisfaction levels, of up to 5 years after surgery of a prospective case series of sixteen patients with single or multiple symptomatic knee-cartilage lesions of modified Outerbridge grades III or IV. Mean baseline age (± SD) was 37 (± 11) years; mean total lesion area (± SD) was 4.4 (± 2.7) cm2. PCs and MNCs were applied intraoperatively on a hyaluronan scaffold. One patient was treated for both knees (n = 17 knees). KOOS Pain, Sport&Rec, and QoL were improved significantly over baseline at 12 months and then again at 3 to 5 years post surgery. KOOS Symptoms and ADL, and the IKDC stayed significantly improved from 12 months to 5 years. All: sphericity-corrected RMANOVA, Bonferroni post-hoc, p < 0.05. Mean ± SD improvements at last follow-up (3-5 years): KOOS Pain 25.5 ± 20.9, Symptoms 27.8 ± 19.0, ADL 26.8 ± 19.2, Sport&Rec 40.0 ± 35.7, QoL 38.8 ± 31.0, and IKDC (in %) 98.9 ± 105.7. Patient satisfaction levels (available for 16 knees): very much, 7; much, 4; mildly, 4; not, 1. Our results indicate that the treatment is clinically effective and mostly satisfactory at midterm. Quantitative biochemical analysis of the repair tissue achieved with single-stage intralesional application of primary autologous articular chondrocytes will be relevant to measure the treatment’s full potential.