BACKGROUND Recently, the clinical additive value of a highly concentrated chlorhexidine (CHX) varnish, when applied subgingivally as an adjunct to scaling and root planing, was described. The objective of the present study was to investigate the microbiological impact of a treatment strategy for chronic periodontitis based on a combination of same-day full-mouth root planing and subgingival CHX varnish administration. METHODS A randomized, controlled, single-blind, parallel trial was conducted on 33 non-smoking chronic periodontitis patients. The control group received oral hygiene instructions and same-day full-mouth root planing. The test group received the same instructions and treatment; however, all pockets also were disinfected using a supersaturated CHX varnish. Subgingival plaque samples were collected from the deepest site per quadrant in each patient at baseline and after 1, 3, and 6 months. Pooled sample analysis was performed using a multiplex polymerase chain reaction-based method for the identification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythensis (Tf), Treponema denticola (Td), and Prevotella intermedia (Pi). RESULTS In terms of detection frequency and bacterial levels, significant, mainly temporary, reductions from baseline were found in both groups. When a comparison was made between the control and the test group, significant differences in the detection frequency of Tf (P = 0.024) and Td (P = 0.024), which favored the test group, were found at 1 month. A similar phenomenon was seen for Td (P = 0.031) based on bacterial levels. An important trend toward lower Tf levels in the test group also was found at 1 month (P = 0.052). Compared to baseline levels, microbiological benefits in the test group seemed to be maintained, at least in part, over a 6-month period. In contrast, all microbial levels had relapsed in the control group at study termination. CONCLUSIONS The microbiological results of the present study promote the subgingival administration of a highly concentrated CHX varnish as an adjunct to same-day full-mouth root planing.