The gold standard for diagnosing invasive candidiasis still relies on blood cultures, which are inefficient and time-consuming to analyze. We developed an in-house qPCR assay to identify the 5 majorCandidaspecies in 78 peripheral blood (PB) samples from ICU patients at risk of candidemia. Blood cultures and (1,3)-β-D-glucan (BDG) testing were performed concurrently to evaluate the performance of the qPCR. The qPCR was positive for DNA samples from all 20 patients with proven candidemia (positive PB cultures), showing complete concordance withCandidaspecies identification in blood cultures, except for detection of dual candidemia in 4 patients, which was missed by blood cultures. Additionally, the qPCR detectedCandidaspecies in six DNA samples from patients with positive central venous catheters blood (CB) but negative PB cultures. BDG values were similarly high in these six samples and the ones with proven candidemia, strongly suggesting the diagnosis of a true candidemia episode despite the negative PB cultures. Samples from patients neither infected nor colonized yielded negative results in both the qPCR and BDG testing. Our qPCR assay was at least as sensitive as blood cultures, but with a shorter turnaround time. Furthermore, negative results from the qPCR provided strong evidence for the absence of candidemia caused by the five majorCandidaspecies.