Escherichia coli are serious pathogens of concern responsible for intestinal and extraintestinal disorders. The presence of antibiotic-resistant pathogenic E. coli in seafood is a growing concern for food safety. This study investigated the antibiotic resistance profile of E. coli (n = 33) representing different pathogroups isolated from seafood. Pathogenic E. coli isolates from fresh seafood samples collected in Western and Southern Mumbai, India, were used for antibiotic susceptibility testing. The Kirby-Bauer disc diffusion method was used for analysing the susceptibility patterns, and the results were interpreted according to the CLSI (Clinical & Laboratory Standards Institute) guidelines. The multiple antibiotic resistance (MAR) index was determined to understand the level of antibiotic resistance. The highest resistance was observed against the third-generation cephalosporins cefotaxime (97%) and cefpodoxime (87.8%), while the least resistance was against chloramphenicol (12.1%) and Co-trimoxazole (18.2%). More than 50% of the isolates were resistant to third-generation cephalosporins, nalidixic acid, ciprofloxacin, aminoglycosides such as gentamicin and amikacin, imipenem, meropenem, piperacillin-tazobactam, Amoxycillin-clavulanic acid, and colistin. The highest (0.95) and the lowest (0.09) MAR indices were recorded for isolates belonging to enterohaemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) pathogroups, respectively. The high resistance to multiple drugs in various pathogroups of E. coli from seafood emphasizes the need to trace and contain the sources of resistant bacteria to ensure the safety of seafood for consumption and prevent dissemination of such strains in the seafood consumer community.