Abstract This study aims to assess in residual periodontal pockets the clinical, microbiological, and local biological effects of antimicrobial photodynamic therapy (PDT), de- livered after ultrasonic instrumentation either once or twice in a 1-week interval. A single center, three-arm randomized longitudinal study was carried out for 6 months. Twenty- eight systemically healthy patients on periodontal mainte- nance with residual pockets (pocket depth (PD) ≥5 mm, clinical attachment loss ≥2 mm, and bleeding upon probing (BOP+)) were included. Residual pockets on three teeth, separated from each other by at least two other teeth, served as study sites. After ultrasonic debridement, they were ran- domly assigned to either PDT delivered twice within 1 week (group A), PDT delivered only once (group B), or sham treatment without activating the laser (group C). Methylene blue was applied with a blunt irrigator tip into the pockets. Sites were irradiated with laser light at a wavelength of 670 nm using a light-diffusing tip introduced into the pocket. Initial PD was 5.9±0.9, 6.3±1.3, and 6.3±1.5 mm in groups A, B, and C, respectively, differences being nonsig- nificant. PD was significantly reduced in all groups. At month 3, PD was significantly lower in groups A (2.9± 1.1 mm; p = 0.04) and B (2.8 ± 1.1 mm; p = 0.03) compared to group C (3.5±1.2 mm). At month 6, none of the sites in group A had persisting pockets PD >4 mm and BOP+, whereas two sites in group B and four sites in group C stayed in this category. Detection frequencies of the studied microorganisms at >1,000 and >100.000 cells/ml did not change significantly from baseline to months 3 or 6 in any group. A significant overall decrease was observed from baseline to month 6 for C-reactive protein, serum amyloid A, fibrinogen, procalcitonin, and α-2 macroglobulin. When looking at the groups separately, C-reactive protein was significantly lower only if the laser had been activated twice (p < 0.05). Other differences between groups were not signif- icant. A single or double episodes of PDT had some additional benefit over ultrasonic instrumentation alone.