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The key objective of current study was to analyse the bacterial diversity of canal water of Lahore, Pakistan. Members of Enterobacteriaceae were targeted that may pose serious health issues to human health.  Total 50 bacterial strains were isolated by using Eosin Methylene Blue agar and MacConkey agar. The 16S rRNA gene sequencing of 30 strains confirmed the final taxonomic status of bacterial strains. The results manifested that strains belong to genera of Enterobacter, Escherichia, Klebsiella, Pseudomonas, Acinetobacter, Aeromonas and Vogesella. Antibiotic susceptibility profile of isolates was evaluated against 13 different antibiotics. E. coli SCE-1 showed highest resistant against majority of the antibiotics. Whereas all strains were sensitive to Amikacin (30 µg). Aeromonas hydrophila SD-6 exhibited highest sensitivity against all antibiotics except ampicillin (10 µg) and amoxicillin (25 µg). None of the bacterial strain was completely resistant or susceptible to all antibiotics. Except E. coli all strains were sensitive to Amikacin (30 µg), Gentamycin (30 µg) and Norfloxacin (10 µg). Most of the strains showed intermediate and resistant antibiotic profile towards Nitrofurantoin (300 µg). Latex agglutination test was performed for the screening of E. coli O157 that showed negative results for this serotype. The detection of potential human pathogens like E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and A. hydrphila in canal water indicates that it is not suitable for domestic purposes, recreational activities, and irrigation