Background: Pediatric patients on chemotherapy are vulnerable to invasive fungal infection especially Candida spices. Resistance to antifungal agents has increased in Candida spp., especially in non-albicans species. This study aims to assess the susceptibility of Candida spp. strains isolated from children with acute leukemia less than 18 years. Materials and Methods: This prospective cross-sectional study was conducted during March 2011 to March 2012. Participants were 188 children aging from 1 month to 18 years, who had acute leukemia, were admitted in Amir Oncology Hospital affiliated to Shiraz University of Medical Science, Shiraz, Iran. Identification of Candida strains was performed using germ tube and chlamydospore production tests on an Application Programming Interface (API) 20 C AUX yeast identification system. Susceptibility testing for 7 antifungal agents was performed by the agar-based E-test method. Fungal cultures were carried out from nose, oropharynx, stool, and urine specimens. Results: A total of 229 yeasts were isolated. C. albicans was the most common species found, followed by C. krusei, C. parapsilosis, C. glabrata, and other Candida species. Candida glabrata was the most highly resistant of the yeasts isolated, being 100% resistant to fluconazole and itraconazole, 88% to posaconazole, and 75% to amphotericin B and ketoconazole. Conclusion: In this study, caspofungin was the most effective antifungal agent against the colonized Candida spp. found, followed by conventional amphotericin B. Knowledge about susceptibility patterns of colonized Candida spp. can be of help to clinicians managing pediatric patients on chemotherapy.