Aim The aim of the study was to accurately assess the antibacterial effect of the combined Er,Cr:YSGG and InGaAsP 940 nm laser therapy on nine pathogenic bacteria in the treatment of periodontitis. Materials and method Fifty-six patients were selected for this pilot study. Five patients were excluded, whereas 51 of them completed the study. The patients were randomly allocated to either the combined 2780 nm Er,Cr:YSGG (Waterlase, Biolase) and 940 nm InGaAsP diode laser (EPIC, Biolase) therapy, adjunct to scaling and root planning (SRP) (experimental group), or to scaling and root planning alone (control group). The quantitative and qualitative analysis of the total number of bacteria and nine specific germs was performed using quantitative real-time polymerase chain reaction. Results The total bacterial load inside the periodontal pockets was reduced both for the laser plus SRP and for the SRP alone group at the 1-month and 6-month follow-ups (p < 0.05). The laser therapy group showed a more significant bacterial reduction than the control group at the 1-month and 6-month follow-ups. The germ number reduction was statistically strongly significant for the total number of germs and for eight out of nine analyzed bacteria. Conclusions The present study suggests that a combined Er,Cr:YSGG 2780 nm and diode InGaAsP 940 nm laser therapy added to the nonsurgical periodontal treatment brings an important benefit in bacterial reduction and stands as a reliable alternative to antibiotic prescriptions in periodontal treatment. The positive changes are also reflected in significant improvement of clinical periodontal parameters. The results suggest that newly formed bacterial microbiome inside the sulcus appears to be more beneficial, durable, and stable in the lased group.