Bone turnover biomarker levels serve as valid tools for assessing bone resorption and formation. While the standard retention protocol consists of using fixed retainers, removable retainers, or both, attempts have been made to explore alternative approaches using biological agents. Psidium guajava fruit is reported to possess osteoanabolic properties. These properties might be useful in preventing orthodontic treatment relapse. To assess the effect of orally administered Psidium guajava fruit suspension on alkaline phosphatase (ALP) and osteocalcin (OC) levels in the gingival crevicular fluid (GCF) of Wistar rats using enzyme-linked immunosorbent assay (ELISA). Fourteen rats underwent orthodontic tooth movement of the left maxillary first molar. Subsequently, the appliance was removed, then retention phase was initiated by placing composite resin in the created space between the left maxillary first and second molars. The intervention group received 250 mg/kg of P. guajava fruit suspension daily, while the control group received normal saline for 35 days. When retention ended, the left maxillary first molars were allowed to relapse for 7 days. At the end of the relapse phase, GCF samples were collected from the distal side of the left maxillary first molars using a PerioPaper and analyzed by ELISA. The mean concentration of ALP in the control and intervention groups was 0.074 ± 0.012 pg/mL and 0.075 ± 0.005 pg/mL, respectively (P = 0.704), while that of OC was 0.696 ± 0.003 pg/mL and 0.692 ± 0.011 pg/mL, respectively (P = 0.391). Oral supplementation of P. guajava fruit suspension did not have a positive impact on GCF bone turnover biomarkers (ALP and OC) in Wistar rats.