The aim of the study was to establish the qualitative and quantitative composition of microbial flora of dental cavities (DCs) after traditional and alternative preparation, including different methods of isolating the working field. Material and Methods: Our study included 60 patients (mean age of 25.0±3.1 years) with DC Class 1 (Black's classification) without concomitant somatic pathology. To accomplish the study’s aim, 60 teeth were prepared. The main group (MG) consisted of 45 teeth prepared under absolute isolation with a rubber dam (RD). In the MG, 15 teeth were treated traditionally with a diamond bur with red and yellow markings (MG-1), 15 teeth were treated by ultrasound with a diamond tip (MG-2), and 15 teeth underwent hydrokinetic preparation (MG-3) with the Aquacut device (Velopex). In the comparison group (CG), which included 15 teeth, DCs were treated traditionally with a dental bur without the RD. The MG and CG were comparable in terms of the initial state of dental and microbiological status. The study of the qualitative and quantitative composition of the DC microflora showed that all DCs contained pathogenic β-hemolytic streptococcus in the CG. At the same time, the maximum number of cases (80%) was moderately contaminated.. MG-1 and MG-2, as in the CG, were characterized by the predominance of β-hemolytic streptococcus at the bottom of the treated cavity. At the same time, the incidence of moderate contamination decreased by 4 times and single contamination increased in the cultures to 80%, compared to the CG (P=0.001). In MG-3, β-hemolytic streptococcus also dominated in the bottom of DCs after RD setting. The number of colonies was single (66.7% of cases) and moderate (33.3% of cases), indicating a significant increase in single and a decrease in moderate infestation, compared to the CG (P<0.01). Analysis of the quantitative characteristics of the microbial composition of the cavity floor after preparation of fissure caries showed the highest bacterial contamination in CG: β-hemolytic streptococcus predominated, averaging 251.20±2.5CFU/tampon. Lactobacillus and Neisseria spp. were detected much less frequently (3.16±1.6 and 1.99±1.3 CFU/tampon, respectively). There was a 10-fold decrease in the number of β-hemolytic hemolytic streptococcus cultures in MG-1 (25.12±2.0 CFU/tampon), MG-2 (25.12±2.0 CFU/tampon) and MG-3 (19.95±2.0 CFU/tampon), compared to the CG (P=0.000). The opportunistic microorganisms in the treatment of hard tissues by different methods (burr, ultrasound, hydrokinetic) under absolute isolation conditions were identified in almost equal numbers, with the Lactobacillus contamination being significantly lower in MG-1, MG-2, and MG-3 than in the CG (P<0.01). Conclusion: After the preparation of DCs, a single presence of opportunistic microorganisms, moderate or single contamination with pathogenic bacteria, and absence of anaerobic bacteria were noted. Absolute isolation with RD provides a reduction of microbial infection regardless of the preparation method, and the maximum positive effect is DC preparation with dental burr and ultrasound.