Invasive infections caused by Candida spp. are increasing worldwide and are becoming an important cause of morbidity and mortality in immunocompromised patients. A large number of manifestations of candidiasis are associated with the formation of biofilms on inert or biological surfaces. Candida spp. biofilms are recalcitrant to treatment with conventional antifungal therapies. The aim of this study was dual 1) to determine the prevalence of biofilm producers among clinical isolates from catheter (16 C. albicans ) and blood culture (2 C. albicans and 30 C. tropicalis), and 2) to determine the activity of amphotericin B and anidulafungin against C. albicans and C. tropicalis biofilms of 24 and 48 hours of maturation. Biofilms were developed using a 96-well microtitre plate model and production and activity of antifungal agents against biofilms were determined by the tetrazolium (XTT) reduction assay. Of catheter and blood isolates, 62.5 and 56.25%, respectively, produced biofilms. By species, 68.42% of C. albicans and 53.33% of C. tropicalis were biofilm producers. C. albicans biofilms showed more resistance to amphotericin B and anidulafungin than their planktonic counterparts. Complete killing of biofilms was never achieved, even at the highest concentrations of the drugs tested. Anidulafungin displayed more activity than amphotericin B against C. albicans biofilms of 24 hours of maturation (GM MIC 0.354 vs. 0.686 microg/ml), but against C. tropicalis biofilms amphotericin B was more active (GM MIC 11.285 vs. 0.476 microg/ml). In contrast, against biofilms with 48 hours maturation, amphotericin B was more active against both species.