This in vitro study examined (a) the anti-bacterial efficacy of a pulsed erbium-doped yttrium aluminum garnet (Er:YAG) laser applied to Streptococcus sanguinis or Porphyromonas gingivalis adhered to either polished or microstructured titanium implant surfaces, (b) the response of osteoblast-like cells and (c) adhesion of oral bacteria to titanium surfaces after laser irradiation. Thereto, (a) bacteria adhered to titanium disks were irradiated with a pulsed Er:YAG laser (λ = 2,940 nm) at two different power settings: a lower mode (12.74 J/cm2 calculated energy density) and a higher mode (63.69 J/cm2). (b) After laser irradiation with both settings of sterile titanium, disks were seeded with 104 MG-63 cells/cm2. Adhesion and proliferation were determined after 1, 4, and 24 h by fluorescence microscopy and scanning electron microscopy. (c) Bacterial adhesion was also studied on irradiated (test) and non-irradiated (control) surfaces. Adhered P. gingivalis were effectively killed, even at the lower laser setting, independent of the material's surface. S. sanguinis cells adhered were effectively killed only at the higher setting of 63.69 J/cm2. Laser irradiation of titanium surfaces had no significant effects on (b) adhesion or proliferation of osteoblast-like MG-63 cells or (c) adhesion of both oral bacterial species in comparison to untreated surfaces. An effective decontamination of polished and rough titanium implant surfaces with a Er:YAG laser could only be achieved with a fluence of 63.69 J/cm2. Even though this setting may lead to certain surface alterations, no significant adverse effect on subsequent colonization and proliferation of MG-63 cells or increased bacterial adhesion was found in comparison to untreated control surfaces.