This study evaluates the potential of erbium, chromium-doped:yttrium, scandium, gallium, and garnet (Er,Cr:YSGG) laser irradiation to modify the titanium surface for optimal seeding of fibroblasts and osteoblasts in the treatment of peri-implantitis. Titanium discs were treated using the Er,Cr:YSGG laser, an ultrasonic device with a stainless tip, or titanium scalers. Changes in surface properties were analyzed by profilometer and scanning electron microscopy (SEM). Murine fibroblast and osteoblast adhesion and proliferation were evaluated qualitatively and quantitatively at 24 and 72 h. Profilometric surface topography and SEM showed that titanium scalers and ultrasonic debridement techniques significantly changed the structure of the machined and rough titanium surfaces. The Er,Cr:YSGG laser irradiation, on the other hand, did not alter titanium microstructures. The Er,Cr:YSGG laser irradiation with the 40 Hz group showed a significantly higher attached fibroblast cell numbers than the titanium scaler group at 72 h after treatment (p = 0.023). Additionally, the number of the attached osteoblasts in the Er,Cr:YSGG laser irradiation with the 40 Hz group was significantly higher than that of the no-treatment groups 24 h after treatment (p = 0.045). The Er,Cr:YSGG laser effectively promoted adherence of fibroblasts and osteoblasts to the titanium surface without significantly altering the titanium surface, suggesting its superiority for treating peri-implantitis.