Fungal infections represent a significant public health challenge due to their high mortality rates, which can be exacerbated by the ability of fungi, especially Candida species, to form biofilms on medical devices. These opportunistic pathogens are causing candidemia in debilitated patients, contributing to increased hospitalization costs and prolonged recovery times. The existing antifungal therapy has some limitations, including high costs, significant toxicity and the emergence of fungal resistance. In this context, drug repositioning and combined therapy may be an alternative. The 8-hydroxiquinoline's derivatives, like nitroxoline (NTX), have shown biological activities, such as antibacterial and antifungal. This study evaluated the antibiofilm potential of NTX, both in its isolated form and in combination, as a new alternative against yeasts and biofilms of clinical interest. The combinations were tested using the checkerboard technique and the drugs were tested against Candida spp. and Trichosporon spp. biofilms at MIC, MICx2, MICx10 and MICx20. The triple combination of NTX, amphotericin B (AMB) and caspofungin (CSP) showed greater effectiveness, with synergic action, against most of the strains. NTX and its combination with AMB and CSP showed excellent action against Candida spp. and Trichosporon spp, being the most effective in reducing the metabolic activities of the biofilm cells. Hence, NTX and its combination showed a promising antifungal and antibiofilm potential, and its repositioning could represent an immediate and safe alternative in the routine of preventing and treating infections in patients using medical devices.