This study aimed to investigate the prevalence, antimicrobial resistance, biofilm forming ability and certain virulence-associated genes of E. coli and P. aeruginosa causing diarrhea and pneumonia in calves and lambs. A total of 101 samples including; 71 deep nasal swabs from pneumonic animals (57 calves and 14 lambs) and 30 fecal swabs from diarrheic animals (19 calves and 11 lambs) were investigated bacteriologically. Regarding pneumonic calves and lambs examined samples, 33 (57.9%) and 7 (50%) E. coli as well as 2 (3.5%) and 6 (42.9%) P. aeruginosa isolates were recovered, respectively. Meanwhile the enteric samples revealed that 17 (89.5%) and 5 (45.5%) E. coli were recovered, respectively while only one P. aeruginosa isolate (5.2%) was recovered from calves. The in vitro antimicrobial susceptibility testing revealed that all E. coli isolates either from pneumonic or diarrheic calves or lambs showed complete resistance against amoxicillin, amoxicillin-clavulanic acid, and ceftriaxone while they showed complete or high susceptibility to amikacin, gentamicin and apramycin. Concerning P. aeruginosa isolates, isolates either from pneumonic calves or lambs revealed complete or even high resistance against amoxicillin, amoxicillin-clavulanic acid, ceftriaxone, cephalexin, kanamycin, and sulfamethoxazole-trimethoprim, while they were completely or highly susceptible to amikacin, apramycin, gentamicin, and colistin. The biofilm formation assay of the tested E. coli and P. aeruginosa isolates from pneumonic samples revealed that a total of 4/9 tested E. coli (44.4%) and 3/6 tested P. aeruginosa (50%) were positive biofilm formers while in enteric isolates, a total of 3/5 E. coli (60%) and one P. aeruginosa from calves (100%) were positive. Results of PCR showed that Five E. coli isolated from diarrheic calves harbored eaeA virulence gene while 60% of the tested isolates harbored hly and chuA genes and 40% harbored yjaA and tspE4C2 genes. The three examined P. aeruginosa isolated from pneumonic lambs harbored fliC, exoS, and toxA virulence genes. In conclusion, the synergism between phenotypic and genotypic methods is effective for characterizing E. coli and P. aeruginosa associated with diarrhea and pneumonia.