In the present work, we addressed the impact of a human-food web-animal interface on the prevalence of food-borne pathogens in mixed farms of Tamil Nadu, India. We have isolated and identified six strains of Clostridium sp. and five strains of Enterococcus sp. from food and animal sources disposed near to the veterinary and poultry farms. Phylogenetic relationships of these strains were inferred from their homologies in 16S rDNA sequences and rRNA secondary structures. The strain PCP07 was taxonomically equivalent to C. botulinum confirmed by neurotoxin-specific PCR primers, followed by mouse bioassay. Other Clostridial and Enterococcal isolates have shown a phylogenetic similarity to the C. bifermentans and E. durans isolated from veterinary farms, respectively. Results of our study revealed that a humanfood web-animal interface has influenced the disease incidence and prevalence of these isolates in the poultry to veterinary farms, where human food acted as a likely transmittance vehicle for their infections.