The past decades have witnessed a rapid increase in the development of resistance to widely used antifungal drugs. Interestingly, interference with iron homeostasis of Candida albicans has been reported to lead to increases fluconazole susceptibility. In yeasts, excess iron is stored in the vacuole, from where it can be released into the cytoplasm during time of scarcity. Genes involved in the transport of iron from the vacuole include FET99 and FTH2. FET99 encodes a multicopper oxidase responsible for the uptake and release of iron stored in the vacuole, while FTH2 encodes a vacuolar iron permease. Importantly, the overall status of the membrane lipids serves as important determinant of drug susceptibility and the relationship between lipid metabolism and iron homeostasis may influence the pathogen’s susceptibility to certain drugs. To explore this interaction, fluconazole sensitivity of Δ/Δ fet99 and Δ/Δ fth2 planktonic cells and developing biofilms was evaluated. These deletions caused a decrease in susceptibility. The lipid droplet, ergosterol content, lipidome and mitochondrial membrane potential of both mutants were also evaluated. It was found that the lipid droplet content was decreased in the mutant biofilms compared to their parental strain. In addition, the mitochondrial membrane, cell membrane and neutral lipidomes of the mutants were significantly influenced, possibly contributing to the decrease susceptibility by limiting fluconazole uptake. This work provides additional information regarding the interplay between lipid metabolism, iron homeostasis and fluconazole resistance in C. albicans, which may be exploited as novel drug targets.