Three cases of buffaloes and their calves were exposed to acute toxicity after feeding on sprayed alfalfa fodder by chlorpyrifos at Monshaat El-Gammal, Tamiyyah city, El-Fayoum Governorate, Egypt.  On treatment of animals with atropine sulphate, adult animals were recovered; two of their calves were recovered within 1 hour, while last one dead. The content of used pesticide active ingredient “chlorpyrifos" and its relevant impurity sulfotep in commercial pesticide formulations (480 g a. i. /L) were estimated by gas chromatography-flame ionization detector (GC-FID) against external standard of high purity. Gas chromatographymass spectrometry method was used for the qualitative and quantitative analyses of chlorpyrifos in the formulated sample and in animal serum, diarrhoeic material as well as milk of exposed animals. The results of analysis showed that the content of chlorpyrifos in the sample is 214.15 g/L which is not comply with the Food and Agricultural Organization (FAO) specifications for the active substance (chlorpyrifos). The content of the relevant impurity sulfotep was estimated as 0.269 % of chlorpyrifos content which comply and close to the corresponding maximum permitted level 3 g/kg of the chlorpyrifos content found. GC-MSMS MRM for chlorpyrifos was 350 m/w for Precursor Ion and Fragment ions were 97, 199 m/z.  The recovery percent of chlorpyrifos ranged between 71.8 and 94.65% with relative standard deviation (RSD) below 1.45%. Average calculated residues of chlorpyrifos were 2.6 ppm in milk, 80.8 ppb in diarrheic materials and 37.4 ppm in grass (alfalfa fodder). This disturbance create suitable media for some pathogenic bacteria invasion, Nine bacterial isolates (4 E. coli, 2 Staph. aureus, 2 Coliform and 1 Klebsiella) from milk, saliva, nasal and faecal samples of 3 buffaloes and 3 buffalo's calves suffering from severe diarrhoea. In conclusion: This study proved that exposure to chlorpyrifos induced disturbance in the intestinal microflora with stimulated pathogenic bacteria causing enteritis. While true used chlorpyrifos pesticiede did not agree with bottle contents. So, more control over pesticides application is required in villages and Ezabs at rural areas.