The genusCandidaspp. has been highlighted as one of the main etiological agents causing fungal infections, withCandida albicansbeing the most prominent, responsible for most cases of candidemia. Due to its capacity for invasion and tissue adhesion, it is associated with the formation of biofilms, mainly in the environment and hospital devices, decreasing the effectiveness of available treatments. The repositioning of drugs, which is characterized by the use of drugs already on the market for other purposes, together with molecular-docking methods can be used aiming at the faster development of new antifungals to combat micro-organisms. This study aimed to evaluate the antifungal effect of diazepam on matureC. albicansbiofilmsin vitroand its action on biofilm in formation, as well as its mechanism of action and interaction with structures related to the adhesion ofC. albicans, ALS3 and SAP5. To determine the MIC, the broth microdilution test was used according to protocol M27-A3 (CLSI, 2008).In vitrobiofilm formation tests were performed using 96-well plates, followed by molecular-docking protocols to analyse the binding agent interaction with ALS3 and SAP5 targets. The results indicate that diazepam has antimicrobial activity against planktonic cells ofCandidaspp. andC. albicansbiofilms, interacting with important virulence factors related to biofilm formation (ALS3 and SAP5). In addition, treatment with diazepam triggered a series of events inC. albicanscells, such as loss of membrane integrity, mitochondrial depolarization and increased production of EROs, causing DNA damage and consequent cell apoptosis.