Introduction Microfragmented adipose tissue (MFAT) has been shown to benefit osteoarthritic patients by reducing pain and supporting tissue regeneration through a mesenchymal stem cell (MSC)-related paracrine mechanism. This observational study of 110 knees assessed patient-centered outcomes of pain, functionality, and quality of life, analyzing their variation at twelve months following one ultrasound-guided intra-articular injection of autologous MFAT for the treatment of knee osteoarthritis (KOA). Method Inclusion criteria were as follows: VAS >50, and the presence of KOA as diagnosed on X-ray and MRI. Exclusion criteria included the following: recent injury (<3 months) of the symptomatic knee, intra-articular steroid injections performed within the last three months, and hyaluronic acid injections prior to this treatment. Changes in VAS, OKS, and EQ-5D were scored at baseline and twelve months following a single intra-articular injection of autologous MFAT. Score variation was analyzed utilizing a nonparametric paired samples Wilcoxon test. The statistical analysis is reproducible with Open Access statistical software R (version 4.0.0 or higher). The study was carried out with full patient consent, in a private practice setting. Results Median VAS (pain) improved from 70 (IQR 20) to 30 (IQR 58) (p < 0.001); median OKS (function) improved from 25 (IQR 11) to 33.5 (IQR 16) (p < 0.001); and median EQ-5D (quality of life) improved from 0.62 (IQR 0.41) to 0.69 (IQR 0.28) (p < 0.001). No adverse events were reported during the intraoperative, recovery, or postoperative periods. Conclusions For patients with all grades of knee osteoarthritis who were treated with intra-articular injections of MFAT, statistically significant improvements in pain, function, and quality of life were reported. Although further research is warranted, the results are encouraging and suggest a positive role for intra-articular injection of MFAT as a treatment for knee osteoarthritis.