The real situation about the role of the small holder's dairy farms and their keepers in maintaining and dissemination of Staphylococcus aureus is fairly unknown. The purpose of this study was to determine the role of animals in transmitting such zoonotic bacteria to their owners owing to study the source of infection. A total of 347 different samples were collected from smallholder's dairy farms at Aziza village, El-Manzala District, Dakahlia Governorate, Egypt in the period between August 2014 and September 2015. The collected samples included 135 animals' fecal swabs (75 from cattle and 60 from buffaloes), 72 cattle milk and 140 human samples (100 stool specimens and 40 hand swabs). In the present study, Staph. aureus was found tremendously in the collected samples (n = 126). For instance: 50% of the tested stool specimens from animal's keepers (n =60) were found to be positive to Staph. aureus. Likewise, 67.5% of keepers hand swabs (n = 40) and 37.5% of diarrheic stool specimens (n =40) were also tested positive. On the other side, out of all recovered Staph. aureus isolates (n = 126), thirty representatives biochemically identified isolates were randomly selected from all examined categories (5 isolates each) and subjected to PCR assay using nuc gene. 2 Only positive samples (n = 25) were further tested by PCR using enterotoxin sea gene. It was found that only six isolates (24%) were shown to harbor sea gene that is responsible for the production of enterotoxin A. From the result herein, it can be concluded that the presence of enterotoxigenic Staph. aureus in cattle feces, buffalo's feces and cow's milk confirm its zoonotic nature and would clarify the role of smallholder's animals as a potential reservoir for Staph. aureus in the examined area. Further epidemiological studies are needed to appraise the role of smallholders in transmitting other zoonotic diseases in a wide scale area.