This study evaluated the influence of Er,Cr:YSGG laser surface conditioning on push-out bond strength of different root posts to the root dentin. Extracted (N = 27, n = 9 per group) and endodontically treated human mandibular premolars were prepared to receive the posts. Three types of posts, namely quartz fiber (D), glass fiber (S), and zirconium dioxide post (C) were luted with resin cement. The posts were randomly assigned to one of the surface conditioning method: (a) No conditioning, control (L0), (b) Er,Cr:YSGG laser at 175 mJ, 3.5 W for 60 s (L1), and (c) at 225 mJ, 4.5 W for 60 s, with 60 μs pulse duration and repetition rate was 20 Hz (L2) irradiation. Six sections (two coronal, two middle, and two apical) were made in each tooth yielding to 1 mm thick specimens. The specimens were stored in distilled water at 37 °C for 24 h and push-out bond strength (MPa) was tested in a Universal Testing Machine (1 mm/min). Data were analyzed using Kruskall–Wallis and Dunns`s post hoc tests (α = 0.05). In group D, both laser treated groups (L1:16.16 ± 19.89; L2:8.24 ± 9.26) presented significantly less bond strength compared to control group (L0:28.3 ± 16.8) (p < 0.001). Mean push-out bond strength values did not significantly differ according to the root segments (coronal, middle, and apical) (p = 0.106). Application of Er,Cr:YSGG laser, with the parameters tested, did not increase the bond strength of zirconium glass fiber and zirconium oxide posts. Laser surface conditioning decreased the bond strength of quartz fiber posts in the root canal.