Background: Candida tropicalis is one of the main non albicans Candida which is implicated in many serious infections. Biofilm formation is an important virulence factor in Candida species. This study was designed to determine the ability of C. tropicalis isolated from infected patients in Pediatric Intensive Care Units (PICUs) to form biofilm, also to test the antifungal susceptibility of Candida tropicalis planktonic cells and biofilms. Methodology: Candida tropicalis isolates were collected from patients suffering from hospital acquired infections in PICUs of Mansoura University Children hospital. The isolates were identified by Analytic Profile Index (API) 20 C. The ability of the isolates to form biofilm was measured by crystal violet assay. The susceptibility of planktonic cells and the biofilms to amphotricin B and fluconazole was determined according to Clinical and Laboratory Standards Institute (CLSI) M27 A2 guidelines and by crystal violet assay respectively. Results : Thirty seven isolates of C. tropicalis were detected during period of study. About sixty percent of the isolates (23 isolates) were biofilm producers. All planktonic cells were susceptible to amphotricin B and 27 isolates (73%) were susceptible to fluconazole. All biofilm cells were resistant to amphotricin B and fluconazole. Conclusion: The biofilm cells expressed higher resistance to the tested antifungal agents more than planktonic cells. Formation of biofilm may represent an important cause of the poor response of infections caused by C. tropicalis to amphotricin B and fluconazole therapy.