Aims: Staphylococcus pseudintermedius is an opportunistic pathogen also indicated as one of the major causes of skin infections in dogs. This study aimed to identify S. pseudintermedius isolated from canine skin lesions, evaluate their antibiotic resistance profile and biofilm production ability. Methodology: Lesions from 50 rural dogs with different skin lesions were sampled after pyoderma diagnosis by private practices. Bacterial species determination was investigated and susceptibility to nine antimicrobials were determined by means of Kirby-Bauer assay. Then seven antibiotic resistance genes, including mecA, blaZ, tetK, tetM, blaSHV, blaOXA-1, and blaTEM were screened by PCR. Moreover, biofilm formation ability of the strains was determined using the microtiter plate assay along with the presence of icaADBC genes. Results: A total of 37 (74%) isolates were identified as S. pseudintermedius. All S. pseudintermedius isolates were resistant to multiple drugs. Resistance to penicillin, amoxicillin-clavulanic acid, and cefazolin were higher than other antimicrobials. All the beta-lactam-resistant isolates carried blaZ, whereas mecA was found in 6 (16.21%) of them. Among tetracycline-resistant strains, the frequency of tetK and tetM determinants were 19 (90.47%) and 21 (100%), respectively. Finally, most cefazolin-resistant strains 31 (91.89%) were positive for blaTEM gene. The rate of biofilm production ability among S. pseudintermedius isolates was 35 (94.59%). Furthermore, the presence of icaA/D genes was detected in 35 (100%) and 29 (82.85%) of S. pseudintermedius strains, respectively. Conclusion: The high rates of antimicrobial resistance and biofilm production ability among S. pseudintermedius isolates, emphasize the urgent need to use more effective infection control policies and guidelines for antimicrobial use.