Introduction: The present study compared the effects of erbium-doped yttrium aluminium garnet (Er:YAG) laser and hand instrumentation on the attachment of human gingival fibroblast (HGF) cells to periodontally involved root surfaces. Methods: A total of 40 tooth specimens were collected and treated in four distinct groups: scaled and root planed with hand instruments, scaled with Er:YAG laser, treated with a combination of hand instruments and Er:YAG laser and non-treated control group. The attachment and proliferation rate of HGF were assessed using MTT assay and scanning electron microscope (SEM) examination was used for cell morphological evaluation. Results: The MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay showed significant decrease in HGF cell viability in both hand instruments only and combination treated teeth specimens compared to control specimens (P < 0.05), 24 hours after cell seeding. However, at time 48, the cell viability of attached cells in these 2 treated groups was almost similar to control. In contrast, at 24 and 48 hours after cell seeding, viability of attached cells was higher than control in Er:YAG laser treated only specimens (P < 0.05). According to SEM study, the laser treated specimens showed more surface roughness. Conclusion: Er:YAG laser increased attachment and proliferation of HGF cells in comparison to the hand instruments method.