Pet shops commonly provide visitors with the opportunity to interact with the animals that are for sale. To assess the risk of zoonotic infection to humans, this study examined the prevalence of bacterial, parasitic and fungal agents in dogs and cats in pet shops in Tokyo. At 54 facilities, we collected 355 fecal and 361 hair coat samples from 364 dogs, and 111 fecal and 112 hair coat samples from 113 cats. Zoonotic agents were detected and included Campylobacter jejuni (5 canine samples), Giardia intestinalis Assemblage A (2 feline samples), enteropathogenic Escherichia coli (O119:NM) (2 feline samples), and Microsporum spp. (4 canine and 4 feline samples). Our study showed that it is important for pet shops to establish a proper quarantine on the premise that pathogens are brought into their facilities, and hygienic control to prevent cross-contamination.