The intensity of heat signs in buffaloes is generally low and the incidence of suboestrus varied from 15 to 73% (Buffalopedia). The objective of this study was to investigate the feasibility of monitoring the changes in some milk constituents, oestradiol levels and electrical conductivity of vaginal mucus during peri-oestrous period in prediction of the timing of oestrus in buffaloes. Twenty-one Egyptian buffaloes aged 3–9 year, 1st–6th lactations, were examined by oestrous detector and ultrasonographically for monitoring the ovarian and uterine activity for 7 days around the time of standing oestrus. Sodium, potassium, chloride and lactose were assayed in aqueous phase of milk; besides, oestradiol was estimated in serum. Current results declared highly significant acute changes in milk constituents at the time of oestrus characterized by peaking of chloride and sodium levels and lowering of potassium and lactose values. The alternation in milk composition when arranged in decreasing order of magnitude, sodium was the highest (77.78 ± 0.69%), followed by chloride (61.60 ± 1.52%) and potassium (−58.14 ± 10.89%). Concomitantly, milk lactose decreased by 26.07 ± 7.97% compared to baseline levels. Synchronously, vaginal electrical resistance (VER) showed a significant (p < 0.01) decrease, but serum oestradiol 17β levels surged (59.93 ± 7.29 pg/ml) on day of oestrus. Serum oestradiol level was negatively correlated with VER (r = −0.577), potassium (r = −0.661), positively correlated with chloride (r = 0.707) and sodium (r = 0.579) and not correlated with lactose levels. These results for the first time suggested that the changes in constituents of milk during peri-oestrous period may be used as a practical non-invasive indicator for oestrous detection and prediction of ovulation in Egyptian buffaloes.