Enterococcus faecalis and Candida albicans are two of the most significant opportunistic and intricate nosocomial pathogens. They reside overlapping niches as major constituents of the gastrointestinal (GI) and oral microbiome. This study was designed to investigate the interaction between these two opportunistic pathogens that could affect treatment strategies and influence the interkingdom signaling and sensing in the microbiome conceptions. E. faecalis was isolated from 150 samples collected from human urine, cattle raw milk, milk products and chicken intestine and identified by both the routine microbiological and molecular identification methods based on 16S rRNA gene. Isolates susceptibility to nine antimicrobials was analyzed using the disk-diffusion method and E. faecalis isolates showed multidrug resistance. Most of C. albicans isolates were able to form biofilm with different intensities. Cell-free supernatant (CFS) of E. faecalis was purified and tested against C. albicans and/or biofilm formation.It inhibited the growth of C. albicans and their filamentation with regards to biofilm formation in vitro. The data presented here demonstrated the potential antifungal activity of E. faecalis CFS and could explain the biological interaction between these two microbes in one hand and the possibility of using E. faecalis CFS as a biological product to control yeast infection in the other hand.