The aims of the study were to evaluate the influence of culture media on biofilm formation byC. albicans, C. glabrata, C. krusei,andC. parapsilosisand to investigate the responses of sessile cells to antifungals and reactive oxygen species (ROS) as compared to planktonic cells. For biofilm formation, theCandidaspecies were grown at different periods of time in YP or YNB media supplemented or not with 0.2 or 2% glucose. Sessile and planktonic cells were exposed to increasing concentrations of antifungals, H2O2, menadione or silver nanoparticles (AgNPs). Biofilms were observed by scanning electron microscopy (SEM) and quantified by the XTT assay.C. albicansformed biofilms preferentially in YPD containing 2% glucose (YPD/2%),C. glabratain glucose-free YNB or supplemented with 0.2% glucose (YNB/0.2%), whileC. kruseiandC. parapsilosispreferred YP, YPD/0.2%, and YPD/2%. Interestingly, onlyC. albicansproduced an exopolymeric matrix. This is the first report dealing with thein vitroeffect of the culture medium and glucose on the formation of biofilms in fourCandidaspecies as well as the resistance of sessile cells to antifungals, AgNPs, and ROS. Our results suggest that candidiasisin vivois a multifactorial and complex process where the nutritional conditions, the human immune system, and the adaptability of the pathogen should be considered altogether to provide an effective treatment of the patient.