Denture stomatitis is associated to Candida albicans, different bacteria and other co-factors such as an acid pH, a carbohydrate ingestion increase, different systemic illnesses and pharmacological treatments. The aim of this study was to determine Candida albicans, Staphylococcus aureus and Streptococcus mutans prevalence in the mucous membrane and prosthesis of patients with and without atrophic denture stomatitis and its relationship with other potential clinical co-factors. Saliva was collected from 105 patients (62 female and 43 male) wearing dental prosthesis in order to measure their pH. Oral samples of the mucous membrane and the internal surface of dental prosthesis were taken with sterile cotton to proceed with the microbiological study. The identification of the isolated microorganisms was performed using conventional microbiological methods. Diabetes and Hypertension were the most frequent systemic illnesses. High carbohydrate ingestion was observed in numerous patients. Atrophic denture stomatitis was reported in 50 patients and the pH average in saliva was of 5.2. The presence of C albicans, S. aureus and S. mutans in the mucous membrane and prosthesis was of 51.4%, 52.4% and 67.6%, respectively. C. albicans was isolated in 66.7% from the prosthesis, whereas S. aureus and S. mutans were isolated in 49.5% of those same prosthesis. C. albicans was isolated in 86% of the patients with atrophic denture stomatitis and S. aureus was isolated in a similar percentage (84% of patients). The isolation of S. mutans was less frequent, and it was observed in 16% of the oral samples of these patients. C. albicans, S. aureus and S. mutans frequently colonize the oral mucous of patients wearing dental prosthesis. This illness-bearing condition is more frequent in patients with denture stomatitis, even though dental prosthesis colonization is lower than in the oral mucous.