The diagnosis of pulmonary candidiasis is still controversial. We undertook a prospective study on 25 non-neutropenic, mechanically ventilated (> 72 h) patients who died in our ICU with the aim of assessing the incidence and significance of the isolation of Candida species from quantitative cultures of immediate postmortem lung biopsies and different respiratory sampling techniques. Immediate postmortem respiratory samples (endotracheal aspirate, protected specimen brush [PSB], bronchoalveolar lavage [BAL], blind biopsies [average 14/patient], and bilateral bronchoscopically guided biopsies [two per patient]) were taken from all patients. Lung tissue specimens were histologically examined. Respiratory samples were classified as having Candida or otherwise. Ten (40%) patients had at least one pulmonary biopsy yielding Candida spp. Among these 10 patients with Candida isolates, only two had definite pulmonary candidiasis. A total of 470 microorganisms were isolated from 280 of 375 (77%) lung biopsy samples in all 25 patients. Candida species represented 9% (n = 40) of the isolates, corresponding to 10 patients (40%). In the 10 patients in whom Candida species was isolated from pulmonary biopsies, this was always associated with the isolation of the same microorganism from one of the sampling methods. Quantitative cultures of Candida species from different sampling methods correlated well among each other but could not discriminate the presence from absence of Candida pneumonia. A logistic regression model adjusted for the presence of antibiotics, days of antibiotic treatment, mechanical ventilation period, age, ARDS, parenteral nutrition, and gender did not show any independent risk factor for developing positive pulmonary samples for Candida species. The incidence of Candida isolation from pulmonary biopsies in critically ill mechanically ventilated, non-neutropenic patients who die is high (40%). However, the incidence of definite Candida pneumonia was 8%. We also found that Candida colonization is uniform throughout the different lung regions, and that the presence of Candida in respiratory samples, independently of quantitative cultures, is not a good marker of Candida pneumonia in critically ill, non-neutropenic, non-AIDS patients.