The objective of this study was to evaluate the microbiological and clinical outcomes following nonsurgical treatment by either scaling and root planing, combination of Nd:YAG and Er:YAG lasers, or by Er:YAG laser treatment alone. The study involved 60 patients with generalized chronic periodontitis, randomly assigned into one of three treatment groups of 20 patients. The first group received scaling and root planing by hand instruments (SRP group), the second group received Er:YAG laser treatment alone (Er group), and the third group received combined treatment with Nd:YAG and Er:YAG lasers (NdErNd group). Microbiological samples, taken from the periodontal pockets at baseline and 6months after treatments, were assessed with PET Plus tests. The combined NdErNd laser (93.0%), followed closely by Er:YAG laser (84.9%), treatment resulted in the highest reduction of all bacteria count after 6months, whereas SRP (46.2%) failed to reduce Treponema denticola, Peptostreptococcus micros, and Capnocytophaga gingivalis. Full-mouth plaque and bleeding on probing scores dropped after 6months and were the lowest in both laser groups. The combination of NdErNd resulted in higher probing pocket depth reduction and gain of clinical attachment level (1.99 ± 0.23mm) compared to SRP (0.86 ± 0.13mm) or Er:YAG laser alone (0.93 ± 0.20mm) in 4-6mm-deep pockets. Within their limits, the present results provide support for the combination of Nd:YAG and Er:YAG lasers to additionally improve the microbiological and clinical outcomes of nonsurgical periodontal therapy in patients with moderate to severe chronic periodontitis.