New alternative laboratory means are needed to improve the options for antemortem diagnosis of avian aspergillosis. In this study, 3-hydroxybutyrate was measured in plasma samples collected from a cohort of African penguins (Spheniscus demersus) maintained under human care. Results were interpreted in combination with those of protein electrophoresis and compared with anti-Aspergillus antibody and galactomannan antigen detection. Overall, 3-hydroxybutyrate levels were found significantly increased in Aspergillus-diseased cases versus the control penguin group (P = 0.002). Mean absolute concentration of β-globulins was increased >20% in samples from infected birds, and α2-globublins were also found to be significantly increased versus clinically normal controls (P < 0.001 and P = 0.001 respectively). Of note, the α2-globulins were also significantly increased versus penguins with inflammatory (non-aspergillosis) diseases (P = 0.001). The specificity of 3-hydroxybutyrate, β-globulins, and α2-globulins for aspergillosis was 78.6%, 79.6%, and 92.2%, respectively. Using these measures in tandem resulted in high specificity (>90%) and negative predictive value (≥80%). In contrast, anti-Aspergillus antibody and galactomannan antigen did not distinguish between infected cases and controls (P > 0.05). This study demonstrates that basic testing in tandem with the new biomarker 3-hydroxybutyrate may provide reliable evidence for the diagnosis of aspergillosis in penguins.