Introduction. Clinically acceptable antiseptic should possess organolithic-mineralolithic properties and antimicrobial efficacy, and should be non-toxic. Objective. The aim of the paper was to assess the presence of genomes of the most common microorganisms (Porphyromonas gingivalis, Agregatibacter actinomycetemcomitans, Tanerella forsythensis, Prevotella intermedia, Treponema denticola and Enterococcus faecalis) in infected tooth root canals before and after rinsing with solution of doxycycline, citric acid and detergent Tween-80 (MTAD) in patients with clinically diagnosed primary apex periodontitis. Methods. The content of primarily infected canals before and after using the MTAD solution was used as a biological material in which the presence of microorganisms DNA was proved. For the detection of bacterial genome the multiplex PCR technique was applied. Results. The percentage of positive samples before canal treatment was 100%. In infected root canals E. faecalis was most dominant (37%). In a relatively high percentage we detected P. intermedia (25%), A. actinomycetemcomitans (20%), T. denticola (17%), T. forsythensis (15%) and P. gingivalis (10%). After rinsing the canal system using MTAD solution, there was a statistically significant decrease in E. faecalis (12%), P. intermedia (0%), T. forsythensis (0%) and P. gingivalis (0%). The presence of other bacteria was also diminished but not statistically significantly. Conclusion. With the application of multiplex PCR technique which provided a simultaneous amplification of various genomic sequences, using several pairs of primers, the most dominant in infected root canals were E. faecalis. P. intermedia, A. actinomycetemcomitans, T. denticola, T. forsythensis and P. gingivalis. After mechanic treatment and irrigation of root canals with MTAD solution, P. intermedia, P. gingivalis and T. forsythensis were not found. The presence of E. faecalis, A. actinomycetemcomitans and T. denticola was diminished, however, not statistically significantly.