Two hundred and fifty-six (256) human faecal samples were collected from thirty-three (33) underground faecal storage cisterns across 5 major towns (Akure, Akungba, Ondo, Ore and Owo) in Ondo State, Nigeria. Salmonella-Shigella agar, Eosine Methylene Blue agar and MacConkey agar, which are selective for faecal bacteria, were used to isolate a total of 103 strains of Gram-negative bacteria. Biochemical characterization of the isolates revealed Escherichia coli (32.04%), Enterobacter aerogenes

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... .56), Klepsiella pneumoniae (12.62%), Salmonella typhi (11.65%), Psudomonas aeruginosa (7.77%), Shigella dysenteriae (6.80%), Proteus mirabilis (5.83%), Citrobacter koseri (3.88%), Providentia alcalifaciens (2.91%) and Klepsiella oxytoca (1.94%). All the bacterial isolates were then subjected to antibiotic sensitivity test using antibiotic discs impregnated with Augmentin (30 µg), Gentamycin (10 µg), Pefloxacin (10 µg), Ofloxacin (5 µg), Streptomycin (10 μg), Sulfomethoxazole-trimethoprim (30 µg), Chloramphenicol (30 μg); Sparfloxacin (10 μg); Ciprofloxacin (5 μg) and Amoxicillin (20 μg). The resistance of the isolates to the antibiotics used varied from 27.18% to Ciprofloxacin, 33.98% to Ofloxacin, 47.57% to Sulphomethazole-Trimethroprim, 56.31% to Streptomycin, 66.99% to Chloramphenicol, 74.76% to Pefloxacin, 75.73% to Sparfloxacin, 75.73% to Gentamycin, 85.44% to Ampicillin and 95.15 to Augmentin. These results showed that underground human faecal storage cisterns contain potential multiple antibiotic resistant bacteria that may be transmitted to human via formites, animal vectors and water.