The study evaluated the reliability of polymorphonuclear (PMN) cell count in endometrial cytology by cytobrush technique as a diagnostic for subclinical endometritis (SCE) at the time of estrus and its relationship with bacterial presence and fertility in buffaloes. Healthy pluriparous buffaloes (n = 115) were subjected to endometrial sampling by cytobrush technique (for cytology and bacterial isolation) and ultrasonography of genitalia prior to insemination at the time of spontaneous estrus. Buffaloes were inseminated at the same estrus and were confirmed for pregnancy by ultrasonography at day 40 post-insemination. Growth of aerobic bacteria was recorded in 26.1% (30/115) of estrual buffaloes. Bacteria of eight distinct genera (Staphylococcus spp., Bacillus spp., Proteus spp., Escherichia coli, Klebsiella spp., Streptococcus spp., Pseudomonas spp. and T. pyogenes) were isolated. Presence of bacterial infection was considered as the standard, against which PMN threshold for SCE was calculated by receiver operator curve (ROC). The PMN count of ≥ 5% with good sensitivity (66.7, 95% CI 50.9-81.4) and specificity (87.0%, 95% CI 78.0-94.6) with an overall high diagnostic accuracy (81.7%) for predicting SCE was obtained. Based on calculated PMN threshold, buffaloes were divided into subclinical endometritic (SCE; ≥ 5% PMNs) and control (< 5% PMNs) groups. Thirty-one buffaloes (27.0%, 31/115) had ≥ 5% PMNs in the endometrial cytology at estrus. Ultrasonographic evaluation showed that size of largest follicle, endometrial thickness, and uterine horn diameter did not differ significantly (P > 0.05) between the two groups. Significantly, lower conception rate (22.58 vs 36.90%, P < 0.05) for first artificial inseminations (AIs) and significantly higher number of AIs per pregnancy (2.81 ± 0.27 vs 1.75 ± 0.15, P < 0.05) were recorded in SCE compared to control group. The results indicated that cytobrush based endometrial cytology with 5% PMN cut-off value has a good accuracy for predicting subclinical endometritis at estrus in buffaloes.