The impact of different acquisition times (AqT) and technetium‐99 m–labeled (99mTc) diphosphonates on the image quality of bone scintigraphy is poorly documented in horses. The aim of this prospective experimental study was to evaluate the impact of varying 99mTc‐disphosphonates and AqT on semiquantitative and qualitative image parameters of bone scintigraphy in horses. Twenty‐four horses undergoing bone scintigraphy were divided equally and randomly into methylene‐ (MDP), hydroxymethylene‐ (HDP), and dicarboxypropane diphosphonate (DPD) groups. Lateral scintigraphic images of the antebrachium were obtained 3 h post 99mTC‐diphosphonate injection using three AqT (60, 90, 120 s). The images were analyzed semiquantitatively using the bone‐soft tissue ratio (B:ST), signal‐to‐noise ratio (SNR), contrast‐to‐noise ratio (CNR), and image contrast. Furthermore, a blinded qualitative analysis was performed using a visual grading analysis. The results showed that DPD images had a significantly higher B:ST ratio than MDP images (P < .01) but not HDP images in all AqT (P > .08). However, DPD and HPD images acquired at 60 s had significantly higher CNR (P < .05) than those acquired at 90 and 120 s. The qualitative analysis revealed no significant differences between 99mTc‐diphosphonates at AqT 60 and 90 s. However, MDP images acquired at 120 s had significantly lower image quality compared to DPD and HDP (P = .01 and .03, respectively). In conclusion, the bone tracers affected the semiquantitative image parameters but not the qualitative analysis findings. Increasing AqT did not necessarily improve the image quality. Therefore, decreasing the AqT enabled a reduction in personnel radiation exposure.