Our objective was to determine the effects of intrauterine infusion of proteolytic enzymes in buffaloes with subclinical endometritis (SCE) at estrus on the resolution of endometrial inflammation and reproductive performance. Buffaloes at spontaneous estrus (E1) were screened for SCE by endometrial cytology to identify SCE (≥5% PMN, n = 22) and non-SCE (<5% PMNs, n = 14) animals. All buffaloes underwent uterine ultrasonographic examination, low volume uterine lavage (cytokines and acute phase proteins) and blood sampling (cytokines and acute-phase proteins) at E1. On the same day (E1), SCE buffaloes were randomly selected either for intrauterine infusion of proteolytic enzymes (ENY, n = 11) or saline (PC, n = 11). Buffaloes without SCE were kept as untreated control (NC; n = 14). All buffaloes were re-examined and re-sampled during subsequent estrus (E2), inseminated during the following estrus (E3), and assessed for fertility related outcomes. Proteolytic infusion resulted a reduction in uterine PMN (P < 0.01) in SCE buffaloes. The concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in uterus, and TNF-α and IL-10 in serum were higher (P < 0.01) at E1 in buffaloes with SCE (PC and ENY) compared to NC. After treatment, uterine IL-1β and TNF-α (P = 0.02), and serum TNF-α and IL-10 were lower within the animals of ENY group (P < 0.01). Before treatment, buffaloes with SCE had higher concentrations (P < 0.01) of serum and uterine amyloid-A and haptoglobin, which decreased (P < 0.01) after treatment in the ENY group. None of the fertility outcomes differ between the treatment groups. In conclusion, intrauterine infusion of proteolytic enzymes reduced endometrial inflammation; however, did not improve reproductive outcomes.