Candida spp. is considered to be the third or fourth most common cause of bloodstream infections associated with healthcare services in the world. Currently, several strains exhibit resistance to the traditional treatments, making the development of new therapeutic molecules necessary. Drug repositioning is an alternative that can be used to work around problems such as toxicity, cost and time in the development of new drugs. This study aims to evaluate the in vitro antifungal effect of tropicamide, molecule of anticholinergic action, against planktonic cells of Candida spp. and biofilm of C. albicans. Six strains of different Candida species were used to determine the minimum inhibitory concentration (MIC) of tropicamide and fluconazole according to CLSI document M27-A3 and one strain of C. albicans was used to evaluate the activity of tropicamide against biofilms. In concentrations of 64μg/mL, the tropicamide exhibited 50% of inhibitory activity in planktonic cell and in concentrations of 128μg/mL is able to inhibit the formation of C. albicans biofilm. Despite the inhibitory activity shown at the present study, the use of a larger number of strains, as well as in vivo cytotoxicity assays, is necessary to confirm the hypothesis that tropicamide can be used as an adjuvant agent in the treatment of infections by the Candida genus.