Infants treated in neonatal intensive care units suffer an increased risk for invasive candidiasis, but the diagnosis is sometimes difficult. D-arabinitol is a metabolite of most pathogenic Candida species. An elevated urine D-arabinitol/L-arabinitol (DA/LA) ratio is a sensitive sign of invasive candidiasis in children with cancer, but the method has not been previously evaluated for newborn infants. We therefore enrolled 117 infants in a neonatal intensive care unit, and 411 urine samples were obtained on filter paper. The DA/LA ratio was measured by gas chromatography-mass spectrometry. For 81 infants with no suspicion of superficial or invasive candidiasis, the urine DA/LA ratio was 2.7 ± 0.7 (mean ± standard deviation [SD]). The upper cutoff level was set at 4.8 (mean plus 3 SD). Of 22 infants with mucocutaneous candidiasis and not given systemic antifungal treatment, two had elevated DA/LA ratios, which normalized after removal of intravascular catheters. Eight other infants were given empiric antifungal treatment but had negative cultures; five of these had repeatedly elevated DA/LA ratios. Six infants with culture-positive invasive candidiasis all had one or more samples with elevated ratios. For seven infants, three with suspected and four with confirmed invasive candidiasis (for which follow-up samples were available), ratios normalized during antifungal treatment. In conclusion, urine DA/LA ratio determination is a rapid test and can be used for newborns. It is possibly more sensitive than fungal blood cultures in the diagnosis of invasive candidiasis and can also be used for monitoring the effect of antifungal treatment.