Experimental studies in animals and in vitro have shown the usefulness of photodynamic therapy (PDT) as an adjunct to periodontal treatment. The aim of this study was to evaluate the long-term clinical and microbiological effects of PDT associated with nonsurgical periodontal treatment. Three sites in each of 33 patients with chronic periodontitis were randomly allocated in a split-mouth design to a treatment group: (1) scaling and root planing (SRP group); (2) SRP and irrigation with toluidine blue O (TBO group); and (3) SRP, irrigation with TBO and low-level laser irradiation (PDT group). Clinical parameters including visible plaque index, bleeding gingival index, bleeding on probing, probing depth, gingival recession and clinical attachment level were measured at baseline, and after 60, 90 and 180 days. Additionally, subgingival plaque samples were collected for microbiological analysis by PCR. Intergroup and intragroup statistical analyses were performed. All treatment groups showed an improvement in all clinical parameters, and a significant reduction in the proportion of sites positive for periodontopathogens at 60, 90 and 180 days compared to baseline (p< 0.05). None of the periodontal parameters showed a significant difference among the groups (p > 0.05). At 180 days, PDT treatment led to a significant reduction in the percentage of sites positive for all bacteria compared to SRP alone (p < 0.05). Within the limits of this study it may be concluded that PDT as an adjunct to periodontal treatment produced statistically significant reductions in some of the key periodontal pathogens but produced no statistically significant benefit in terms of clinical outcome.