Background: With laser irradiation emerging as an adjunctive treatment utilized in nonsurgical periodontal therapy, it is important to understand the variance of penetration depth among the different laser wavelengths. Purpose: To evaluate the thermal penetration depth, as a photothermal effect, of carbon dioxide (CO2)-, erbium: doped yttrium-aluminum-garnett (Er:YAG)-, and erbium, chromium: yttrium-scandium-gallium-garnett (Er,Cr:YSGG)-lasers on the bovine gingiva in an in vitro model. Methods: Four mandibles from freshly slaughtered cows were utilized in this study. Buccal and lingual root debridement was provided using three different laser wavelengths, all in pulsed settings. A CO2- (10,600 nm), Er:YAG- (2940 nm), and Er,Cr:YSGG- (2780 nm) were utilized to irradiate pockets of two mandibular posterior teeth in each group. Laser power output settings were set to 2 W. The posterior teeth were irradiated for 30 sec buccal and 30 sec lingual of each tooth for all selected treatment test groups. Instrumentation with curettes was performed as a control group. Gingival flaps, including the entire gingiva, were fixed in 10% formalin and stained via Elastin van Gieson. Sections were examined microscopically to evaluate thermal damage and statistically compared using mixed effect model with Tukey adjustment. Results: The CO2-laser irradiation presented a statistically significant lower mean compared to Er,Cr:YSGG-laser (p < 0.0001). Er,Cr:YSGG-laser had a higher penetration depth compared to Er:YAG-laser (p < 0.0001). There was no statistically significant difference found in penetration depth between CO2- and Er:YAG-laser irradiation. Conclusions: It can be concluded that all tested pulsed lasers had minimal penetration depth into the gingiva. However, the pulsed CO2- and Er:YAG-lasers presented lower thermal effects compared to Er,Cr:YSGG-laser in vitro.