Introduction <p>VirClia^® provides an automated platform for detecting antibodies against <i>Bartonella henselae</i> and <i>Coxiella burnetii</i>.</p> Objective <p>To assess the diagnostic performance of VirClia^® and identify potential sources of interference.</p> Methods <p>A retrospective study was conducted using two independent datasets from routine clinical samples tested simultaneously by IFA and VirClia^®. Diagnostic performance was evaluated using Cohen’s kappa and ROC curves, with IFA as the reference standard. For <i>C. burnetii</i> (<i>n</i>=1628), IgG against Phase I, Phase II, and their combination (positive if either reactive) was analyzed. For <i>B. henselae</i> (<i>n</i>=955), IgG and IgM were assessed independently.</p> <p>Two thresholds were defined per parameter: one ensuring 90% sensitivity and another ≥90% specificity. Intermediate values were considered within a gray zone, assumed to require IFA confirmation. The potential reduction in IFA use was estimated assuming that values outside this zone would not need confirmation.</p> <p>False positives (FP) were defined as VirClia^® values above the high-specificity threshold with a negative IFA result. Logistic regression was used to assess associations between FP results and sex, age, and symptoms. The presence of autoantibodies in FP cases was also evaluated.</p> Results <p>Applying gray zone thresholds—using the manufacturer’s upper cut-off (1.1) and a sensitivity-driven lower limit—yielded estimated IFA reductions of 56% for <i>B. henselae</i> IgG, 42% for IgM, and 79% for <i>C. burnetii</i> (combined phases). FP results for <i>C. burnetii</i> were significantly associated with extractable nuclear antigens and older age.</p> Conclusion <p>VirClia^® demonstrates strong diagnostic performance and may reduce IFA dependency when a gray zone strategy is implemented.</p>