Pseudomonas aeruginosa and Klebsiella spp. are opportunistic pathogens linked to several diseases in both humans and animals, posing a major risk to both human health and the advancement of animal husbandry. The present investigation aimed to determine the prevalence of such pathogens in sheep, cattle, and humans as well as to assess the antimicrobial sensitivity pattern in relation to the most widely prescribed antibiotics and, additionally, to use molecular assays for illustrating the genes linked to biofilm and virulence.  Fecal samples were gathered from 791 cattle and 386 sheep, beside 304 human stools. Significant isolation of Pseudomonas aeruginosa was recorded in 16.81%, 7.51%, and 5.59% of cattle, sheep and humans, respectively, while Klebsiella pneumoniae was found in 1.8%, 8.9%, and 1.97% of the cattle, sheep, and human samples. Likewise, Klebsiella  oxytoca was isolated from 2.91%, 4.4%, and 0.33% of the samples. The majority of the isolated bacteria showed inconsistent patterns of resistance to different antibiotics, however, ceftriaxone showed favorable effects. The molecular characterization of the tested isolates showed that they were virulent and revealed gene determinants for the ability to form biofilms (pelA and rmpA). It was clarified that antibiotic resistance will lengthen the course of treatment because biofilm formation in the isolates prevents antibiotics to reach  the bacteria. Furthermore, there was a correlation between the emergence of multi-drug resistance and elevated biofilm production.