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Though mastitis is caused by multiple etiological agents, bacteria are the most common etiological agents. The molecular detection of an organism is less time-consuming and more accurate than conventional methods, hence the present study was aimed at conventional as well as molecular detection and the antibiogram study of E. coli from the suspected subclinical (15) and clinical (15) mastitis milk samples collected in Puducherry’s Aryankuppam region. All the collected samples were positive for the California Mastitis Test. Subsequently, 19 E. coli isolates were isolated, and they were investigated using the PCR technique (uspA gene) as well as biochemical tests. Twelve samples were found positive by PCR (uspA gene). Furthermore, an antibiogram study revealed that penicillin (100%) was highly resistant, followed by nalidixic acid (78%). Notably, the highest susceptibility was observed for ciprofloxacin (92%), followed by gentamicin (85%), and ceftriaxone (71%). The probable isolates obtained need to be additionally investigated using more mastitis milk samples. [ABSTRACT FROM AUTHOR]