Discrepancies in results between cerebrospinal fluid (CSF) 14-3-3 protein and prion protein detection using real-time quaking-induced conversion (RT-QuIC) might limit the confidence in ante-mortem clinical diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). We aimed to evaluate the concordance and diagnostic performance of 14-3-3 protein and RT-QuIC in a real clinical practice cohort, and to analyze neuronal/glial damage biomarkers in sCJD patients based on their diagnostic assay results. A retrospective multicentre study was conducted on 157 suspected sCJD patients from 38 Spanish hospitals in a 4-year period. CSF 14-3-3 protein and RT-QuIC were simultaneously evaluated in a single laboratory. A diagnosis of probable sCJD was established in 63 patients (40.1%), of which 12 (19.0%) were ultimately diagnosed with definite sCJD. Forty-one sCJD patients (65.1%) were positive for both 14-3-3 protein and RT-QuIC, 17 (27.0%) isolated positive for RT-QuIC, and 5 (7.9%) isolated positive for 14-3-3 protein. RT-QuIC demonstrated higher sensitivity (92.1%) and specificity (98.9%) compared to 14-3-3 (73.0% and 62.8%) for the diagnosis of sCJD. Isolated RT-QuIC positivity was associated with longer disease duration (median: 10.5 months, IQR: 8.8-15.7), higher frequency of Met/Val cases (75.0%), lower prevalence of periodic sharp-wave complexes (5.9%), and lower levels of GFAP (3967.4 pg/mL), UCH-L1 (2218.1 pg/mL), and t-Tau (228.8 pg/mL) compared to double-positive and isolated 14-3-3-positive patients. In conclusion, CSF RT-QuIC is a highly specific and sensitive biomarker for ante-mortem sCJD diagnosis and should be considered as the preferred CSF ancillary test. Isolated RT-QuIC positivity indicates a less aggressive biological disease in sCJD patients.