Background The aim of this trial was to evaluate the cytokine, chemokine, and growth factor levels in peri-implant sulcular fluid (PISF) during healing and osseointegration at osteotomy sites prepared either with piezosurgery (PS) or drills. Methods Fourteen patients having contralateral partial edentulism in the posterior maxilla were enrolled and 38 osteotomies were prepared. Implants were placed with one-stage surgery. Insertion torque, early healing index, probing depth and modified gingival and plaque indices and crestal bone loss (CBL) were measured. PISF was collected from each implant at weeks 2, 4, 8, 12, and 24 and were analyzed by a 30-Plex immunoassay. Data analysis employed Brunner-Langer method. Results CBL values did not depend on osteotomy modality (P > 0.05). Eighteen molecules (interleukine (IL)-1 beta, granulocyte colony stimulating factor (G.CSF), IL-13, IL-6, IL-12, interferon (IFN)-gamma, IFN-alpha, IL-2, IL-2 R, IL-8, macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, monocyte chemoattractant protein (MCP)-1, interferon gamma-induced protein (IP)-10, monokine induced by IFN-gamma (MIG), epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) showed time-dependent decrease (P < 0.05), but they were not treatment-dependent (P > 0.05). When values of weeks 4, 8, 12, and 24 were compared to week 2, it was found that all were highest at week 2 and decreased thereafter (P < 0.05). The decrease was significant at weeks 4 or 8 for multitude of molecules and was mostly sustained throughout the follow-up. Week 8 regulated on activation, normal T cell expressed and secreted (RANTES) values in PS group were lower in PS group compared to drill group (P < 0.05). Conclusions Implants placed into osteotomies created with PS and drills are similar in terms of PISF biomarker changes during the osseointegration and wound healing period. When clinical and CBL parameters were taken into account together with the PISF molecular data it can be speculated that PS and conventional drill osteotomy have similar effects on peri-implant tissues on the biochemical, clinical and radiological levels.