Background and Aim: The reproductive performance of dairy cows is of a high economic value to maintain efficient and sustainable productivity. Postpartum anestrus is one of the major infertility problems that cause limitation of dairy cow reproductive performance and productivity. The previous studies have reported using a polyherbal formulation for different purposes, including reproductive performance. This study was conducted to examine the efficacy of polyherbal formulation administration on the reproductive performance of cows and to establish the oral administration of polyherbal formulations as a safe, effective, and economic treatment for dairy cows with postpartum anestrus due to nutritional disorders, negative energy balance, high milk production, and/or heat stress. Materials and Methods: A total of 14 dairy cows with postpartum anestrus were randomly divided into two groups, which were subjected to oral administration of distilled water that served as the control (Ctrl; n=5) or polyherbal treatment (polyherbal treated; n=9) for two shots of treatment (each for 3 successive days) with a 10-day interval. Blood sampling and ultrasonography were performed before treatment and after the first and second shots of treatment. Progesterone (P4) assay was also performed. Results: The estrus induction rate was 66.7% after the first shot of treatment in the polyherbal-treated group, which increased significantly to 88.9% after the second shot of treatment, compared with the Ctrl group (20%). The estrus response was confirmed by ultrasonography and P4 hormone assay, wherein the polyherbal treatment significantly increased the P4 concentration in the polyherbal-treated group after the second shot of treatment compared to that before treatment and in the Ctrl group at all time points. On pregnancy diagnosis, the treated dairy cows showed conception rates of 66.7% and 20.0% in the polyherbal-treated and Ctrl groups, respectively. In contrast, the concentration of malondialdehyde, an oxidative stress marker, and the total antioxidant capacity remained unchanged between both groups before and after treatment. Conclusion: The polyherbal formulation containing tubers of C. rotundus, M. pterygosperma, rhizome of Z. officinale, and A. cepa has the potential to induce estrus response and luteal activity in dairy cows and is a possible treatment for ovarian inactivity in dairy farms.