One hundred fifty samples (raw bovine milk, milk from mastitis bovine, teat swabs, swabs from milkers hands and urine from pregnant women) were collected from Basrah. Bile-Esculin agar and biochemical tests were used for isolation and identification bacteria , the diagnosis was confirmed automatically by VITEK 2 system. Drug susceptibility testing was performed for seven antibiotics by CLSI criteria . Biofilm production was quantified by microtiter plate . The results showed 40 Enterococcus isolates with a prevalence 26.6% (30 E. faecalis and 10 E. faecium). All the isolates were 100% resistant to ampicillin and high rates resistance to tetracycline (62.5%) and ciprofloxacin (57.5%) . On the other hand, the isolates showed highest sensitivity to linezolid (62.5%) then vancomycin (50%). Most of the isolates (72.5%) were classified as weak biofilm producers and two isolates were classified as moderate . The mean biofilm production was highest in urine isolates (0.060 ± 0.028) and lowest in hands isolates (0.042 ± 0.006). Similarly, E. faecalis was also a better biofilm producer than E. faecium with an average 0.050 ± 0.018 versus E. faecium (0.045 ± 0.010). The study showed high prevalence of multidrug resistance isolates with complete resistance to ampicillin being the commonest. The results also show that clinical or microbial environment (the source of the isolates) has a transmission pressure that clearly influences the variation of resistance ratio and the capacity for biofilm formation, highlighting the importance of improved health surveillance to restrict transmission of these pathogens between bovine and human environments.