Denture-related stomatitis (DRS) is still a dilemma in removable prosthodontics. The aim of this study was to investigate the relationship of DRS with the presence of Candida albicans hyphae and Lactobacillus. A total of 91 patients wearing maxillary and mandibular complete dentures were included in the present study and tested mycologically as well as bacteriologically. A statistically significant association of DRS was found with denture age (p = 0.003) and continuous denture wearing (p = 0.015). Presence of C. albicans hyphae was shown to be significantly higher in DRS cases (p < 0.01), and there was a statistically significant positive correlation between presence of hyphae and C. albicans (p < 0.01). Another interesting finding was that DRS patients showed higher Lactobacillus counts in their saliva (p = 0.04), as well as in the palate (p = 0.028). C. albicans is an important factor in the development of DRS. Hyphae seem to facilitate the rise of C. albicans counts and be related to the inflammatory response of the tissues. Lactobacillus seems to play an important role in the presence of DRS, as well. In agreement with many other studies, the results of this study confirm the importance of denture age and continuous denture wearing in the development of DRS.