The recognition that destructive periodontal diseases may be caused by specific microorganisms in periodontal pockets has led to an increased interest in and usage of antimicrobial agents in periodontal therapy. Recently, a new controlledrelease insert containing ofloxacin, a synthetic antibiotic, has been developed. In this study, the controlled‐release insert (PT‐01) was microbiologically evaluated in combination with or without subgingival mechanical debridement. PT‐01 was applied in the periodontal pockets of 27 patients with chronic periodontitis. Three sites with a deep probing pocket depth (≥5 mm) were randomly selected in different quadrants of each patient, and were assigned into three groups, i.e., PT01 applied (T), placebo applied (P) and control sites (C). Periodontal treatments consisted of supragingival scaling with oral hygiene instruction for the first 2 weeks followed by root planing and subgingival scaling. PT‐01 was applied weekly on day 0 to 35, and the subgingival plaque samples from each site were collected on d 0, 14, 21 and 42. The dynamics of the subgingival microflora was investigated by dark field microscopy and by anaerobic and aerobic cultivation. In the supragingival scaling period, significant reduction in percentages of spirochetes and motile rods and significant increase of the percentage of coccoid cells were observed only at T sites. In addition, the total viable counts of bacteria, black‐pigmentedBacteroidesandFusobacteriumspecies were significantly reduced at T sites. After mechanical subgingival debridement, significant shifts in the proportion and reduction of the viable counts in the subgingival microflora were found at all sites. Although PT‐01 applied sites showed some further shift in the proportion and reduction in subgingival microorganisms, statistically no significant differences in the microbiological results between the three groups of experimental sites were found. These results suggested that weekly application of PT‐01 in the periodontal pocket could have significant effects on both qualitative and quantitative changes in the subgingival microflora. However, the mechanical subgingival debridement, consisting of root planing and subgingival scaling, was very effective in eliminating the subgingival microflora by itself. Then, in the combination with mechanical subgingival debridement, statistically no additional effects could be found. Consequently, it was suggested that the application of PT‐01 might have an ameliorating effect as an adjunct in conventional pe