BACKGROUND: Complete dentures are a valid treatment option in edentulous patients. However, the average service life of complete dentures is 4–5 years, which is frequently due to microbial contamination. AIM: To perform quantitative and qualitative assessment of microbiota on the surface of complete dentures after 1 and 4 years of use. MATERIALS AND METHODS: The study included 40 fully edentulous patients (К08.1) who used acrylic complete dentures for no more than 5 years. There were two groups (n=20 each) based on the duration of use of complete dentures: 1 year in Group 1 and 4 years in Group 2. The microbiota composition was examined by mass spectrometry. The Statistiсa 13 package was used for statistical processing of the study findings. For multiple comparisons, the parametric t-test with Bonferroni correction was used to assess intergroup differences, with p=0.05 as the critical significance level. RESULTS: Microbial contamination increased in all examined patients after using complete dentures for 1 to 4 years. Cocci and bacilli counts increased from 56±5 (105 cells/g) (р=0.03) to 107±8 (105 cells/g) (р=0.04). Anaerobe counts increased from 68±6 (105 cells/g) (р=0.0002) to 102±9 (105 cells/g) (р=0.0002). Actinobacteria counts increased from 30±3 (105 cells/g) to 143±12 (105 cells/g) (р=0.003). Gram-negative rod counts increased from 4±1 (105 cells/g) (р=0.0005) to 24±2 (105 cells/g) (р=0.0006). Yeast and mold counts increased from 977±90 (105 cells/g) (р=0.0003) to 1,587±136 (105 cells/g) (р=0.003). CONCLUSION: Within 4 years, yeast and mold counts increased by 91%, actinobacteria counts by 61%, gram-negative rod counts by 500%, and anaerobe counts by 50% in all patients. The study findings indicate that microbial contamination of dentures is directly related to the duration of their use.