This study aimed to assess the in vitro antimicrobial effects of chlorhexidine (CHX) and povidone-iodine (PI) on clinical isolates of Escherichia coli (E. coli) and Trueperella pyogenes (T. pyogenes) from the vaginal discharge of dairy cows, as well as to compare the cytotoxicity effects of CHX and PI on bovine endometrial epithelial cells (BEnEpC). In Experiment 1, 12 E. coli and 10 T. pyogenes were isolated from the vaginal discharge of cows with a uterine infection. The MIC and MBC against CHX and PI were analyzed in vitro. In Experiment 2, the cytotoxicity effects of CHX and PI on BEnEpC were analyzed using a Viability/Cytotoxicity Kit, wound scratch healing assay, and the expression of pro-inflammatory cytokine genes (IL-6, IL-8, and TNF-α). In Experiment 1, the MIC and MBC values of CHX against E. coli were 0.0002% and 0.0002 to 0.00025%, respectively. The MIC and MBC values of PI were 1.25 to 2.5% and 1.25 to 5%, respectively. For T. pyogenes, the MIC and MBC values of CHX were 0.00002%. The MIC and MBC values of PI were 1.25%. In Experiment 2, the cell viability significantly decreased, and wound closures were significantly inhibited after treatment with ≥ 0.002% CHX and ≥ 0.025% PI. The expression of IL-6, IL-8, and TNF-α significantly increased after treatment with PI. Only IL-6 showed a significant increase after cells were treated with 0.00002% and 0.0002% CHX. The results suggested that both CHX and PI had high antibacterial effects. However, veterinarians and farmers should be aware of their cytotoxicity, which decrease viability of endometrial epithelial cells and inhibit wound healing in vitro.