Several yeast isolates can secrete antifungal components. These include toxic proteins or enzymes, cyclic dipeptides, surfactants, or volatile small molecules, all of which act against fungi. Although the term 'killer yeast' traditionally refers to those that secrete protein toxins, some literature uses the term more broadly for any yeast with antifungal activity. This study aims to obtain highly potent killer yeast isolates to be used in biological control against several other microorganisms. 30 yeast isolates were collected from three distinct sources (soil, juice, and fruit) from seven different locations: Assiut town, Bosra, Sahel Selim, Elbadari, AlGhanium, Abnob, and Manfalout, all located at Assiut goernorate, Egypt. The isolates were characterized and tested for their killing activity. Five isolates out of 30 showed killing activity. ITS gene sequence determination method was used to identify the strongest isolate molecularly, and the results showed that isolate no. 9 was 99% related to Meyerozyma carbibica. The potential of the tested isolates to eliminate various pathogens was demonstrated by their antagonistic against several plant pathogenic microorganisms. The results showed the ability of the tested isolates to kill different pathogens. Therefore, these isolates can be used to biologically control various plant pathogens.