Mastitis is caused by many pathogens including Staphylococcus, Streptococcus, Escherichia, Mycoplasma, etc. Environmental mastitis is majorly caused by E. coli. Aim of the study was to detect toxigenic genes and antibiotic resistance of invasive E. coli. Milk samples (n= 150) were screened for mastitis by California mastitis test (CMT). The CMT positive samples (n=100) were processed. Eight isolates were identified as E. coli. Then multiplex PCR was performed for detection of toxigenic genes. All isolates were positive for toxigenic genes including aggR (100%) followed by est and elt (62%), eae (37.5%) and Asp (25%), respectively. Antibiotic susceptibility test was performed using different antibiotics. Isolates were resistant to Cefixime (37%) followed by Ampicillin, Ceftriaxone and Co-trimoxazole (25%), Amoxicillin, Tetracycline, Nalidixic acid and Cefotaxime (12%), respectively. It was concluded that eae, elt, est, aggR and Asp genes were present in E. coli isolates and highly resistant to Cefixime, minimum resistant against Gentamicin and Ciprofloxacin.