Description: Background: The yeast Candida is one of the most frequent pathogens isolated from bloodstream infections and is associated with significant morbidity and mortality. Problems with clinical and microbiological diagnosis of invasive candidiasis (IC) have prompted the development of non-culture-based laboratory methods. Previous reports suggest that serological detection of antibodies might be useful for diagnosing systemic candidiasis. Methods: Diagnosis of IC using antibodies against recombinant Candida albicans enolase (Eno) and fructose-bisphosphate aldolase (Fba1) was evaluated. Using recombinant Eno and Fba1 as coating antigens, enzyme-linked immunosorbent assays (ELISAs) were used to analyze sera from patients with candidemia (n = 101), Candida colonization (n = 50), bacteremia (n = 84), invasive aspergillosis (n = 40); and from healthy controls (n = 200). Results: The results demonstrated that ELISA detection of anti-Eno and anti-Fba1 IgG distinguished IC from other pathogenic infections in patients and healthy individuals. The sensitivity, specificity, and positive and negative predictive values were 72.3%, 94.7%, 78.5% and 93% for anti-Eno, and 87.1%, 92.8%, 96.4% and 76.5% for anti-Fba1 antibodies, respectively. Combining these two tests improved sensitivity up to 90.1% and negative predictive value up to 97.1%, with specificity and positive predictive values of 90.6% and 72.2%. The tests were specific to the Candida genus and antibody titers were higher for candidemia patients than for controls. Positive antibody tests were obtained before blood culture results for 42.2% of patients for anti-Eno and 51.1% for anti-Fba1. Conclusion: These data suggest that tests that detect IgG antibodies against Candida enolase and fructose-bisphosphate aldolase, especially when used in combination, could be a powerful tool for diagnosing IC.