This study aims to evaluate the effects of 940 nm diode laser and 2780 nm erbium, chromium-doped: yttrium, scandium, gallium, garnet (Er,Cr:YSGG) laser used in addition to mechanical therapy in the non-surgical treatment of peri-implantitis on clinical parameters and matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels in the peri-implant crevicular fluid. A total of 50 patients with peri-implantitis were randomized into three groups to receive peri-implant treatment. The control group (n = 17) only received conventional non-surgical mechanical therapy. The trial groups [(diode group (n = 16) and Er,Cr:YSGG group (n = 17)] received dental laser in addition to mechanical therapy. Gingival index (GI), plaque index (PI), bleeding on probing, probing depth (PD), MMP-9, and TIMP-1 levels were assessed at baseline (T0) and at 6 months after treatment (T1). The GI, PI, and PD significantly decreased in all groups at T1, compared to T0 (p < 0.05). The decrease in the PD was similar between the control and diode groups with Er,Cr:YSGG providing more reduction (1.16 +/- 0.64 mm) than either method (p = 0.032). A significant intra-group decrease in MMP-9 level was only observed in the Er,Cr:YSGG group (p = 0.009). The decrease in TIMP-1 level from T0 to T1 was similar between the control and the diode groups (p > 0.05) and it was significantly lower than the decrease in the Er,Cr:YSGG group (p < 0.05). Addition of diode laser to non-surgical mechanical therapy does not provide any additional benefit for treatment outcomes. The Er,Cr:YSGG laser seems to be more efficient both at clinical and molecular levels.