Recent studies have shown that bisphosphonates can also impact osteoblasts besides osteoclasts. This study aimed to evaluate the effects of different concentrations of Zoledronic acid (ZA) during the osteogenic differentiation of human Bone Marrow Stem Cells (hBMSCs) in vitro. Thus, osteogenic differentiation of hBMSCs was conducted with different concentrations of Zoledronic Acid (ZA) (0, 0.1, 1.0, and 5.0 μM) for the first 3 days. Cell metabolism was quantified at 1-, 3-, 7-, and 14 days. At 7- and 14-days, the following analyses were performed: 1) mineralization nodule assay, 2) LIVE/DEAD™, 3) cell adhesion and spreading, 4) alkaline phosphatase (ALP) activity, and 5) qPCR analysis for RUNX-2), ALPL, and COL1 A1. Data were analyzed by ANOVA 2-way, followed by Tukey's post hoc test (p < 0.05). Cell metabolism (3-, 7-, and 14-days) (p < 0.001), mineralization (7-, 14-days) (p < 0.001), and ALP activity (14-days) (p < 0.001) were reduced in ZA 5.0 µM when compared to control (no ZA). Also, ZA 5.0 µM downregulated the expression of RUNX2 at 7- and 14-days (p < 0.001). It is possible to conclude that ZA (5.0 µM) can impair hBMSC differentiation into osteoblasts and interferes with its mineralization phase.