This study aims to evaluate the prevalence of multidrug-resistant (MDR) and biofilm-forming pathogens from animal source compared to clinical ones. In addition, to assess the antibacterial and antibiofilm activity of silver nanoparticles (AgNPs) alone and/or mixed with vancomycin. Out of 62 bacterial isolates from animal respiratory tract infection (RTI), 50.00% were defined as MDR, while among human ones, 44.00% were MDR. The bacteria Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae were the predominant isolated bacteria from both animal and human origin with frequency percentage of 50.00, 22.32, and 18.75, respectively. Among Staph. aureus strains, mecA gene was detected in 60.00% and 61.54% of animal and human isolates, respectively, while mec ALGA251 (mecC) gene was detected in 13.33% and 15.38% of animal and human isolates, respectively. Biofilm formation ability among animal isolates was 83.87%, while among human ones was 86.00%. AgNPs were effective in inhibiting planktonic cells with minimal inhibitory concentration (MIC) values (0.625-10 mg/mL), as well as eradicating biofilm with minimal biofilm eradication concentration values (1.25-10 mg/mL). Noticeable low MIC of AgNPs was required for the isolates from animal source (0.625-5 mg/mL) compared to clinical ones (0.625-10 mg/mL). Remarkable reduction in AgNP effective concentration was observed after combination with 1/4 MIC of vancomycin with minimum recorded concentration of 0.08 mg/mL. In conclusion, the prevalence of MDR among RT pathogens was recorded with high ability to produce biofilm and virulence factors from both animal and human pathogens. AgNPs showed strong antibacterial and antibiofilm activity alone and mixed with vancomycin, with up to fourfold reduction of AgNP inhibitory dose.