Neopterin and soluble CD14 (sCD14) are detected at high levels in hepatitis C virus (HCV) infections. We aimed to evaluate the role of these plasma immune activation biomarkers, for the indirect assessment of immune activation status of patients with low anti-HCV reactivity and a HCV infection. Low anti-HCV reactivity group (LRG, n: 70), true positive HCV infection group (THG, 30) and healthy control group (HCG, 30) were analyzed in this study. We have used ELISA, HCV RIBA/LIA and HCV-RNA methods. Mean neopterin levels were significantly lower in LRG than THG (p <0.001). In contrast, those values were not significantly different from those of HCG (p >0.05). Mean sCD14 were significantly higher in LRG than THG and HCG (p <0.05, p <0.001). Values of 3.95 mu g/ml and 5.36 nmol/l for sCD14 and neopterin resulted in the maximum area under the receiver operating characteristic curves (ROC), which were 0.859 (95% CI, 0.745 to 0.935; <0.0001) and 0.788 (95% CI, 0.663 to 0.883; <0.0001), respectively. These cut-offs corresponded to a sensitivity of 73.3% and a specificity of 73.3% for neopterin and of 100% and 76.7% for sCD14. Our results suggest that a specific immunoactivation might be caused by true positive HCV infection. Due to the significant results sCD14 in LRG might be non-specifically affected by some underlying atypical immunohematological pathologies. Only neopterin might be used to exclude low anti-HCV reactivity from a true HCV infection. The use of neopterin but not sCD14 in combination with fourth-generation EIA/CMIA combo tests will be useful when nucleic acid tests are not available for screening blood donors at blood banks.