Resistant infection with multidrug-resistant Escherichia coli (E. coli) is one of the most common causative agents of bacterial infections and emergence as a multidrug resistant bacterium is a major public health threat worldwide and representing a real problem for health care providers. The use of Bacteriophage lytic enzymes (lysins) are highly evolved molecules that have been specifically developed by phages to quickly and efficiently allow their progeny to be released from the host bacterium while destructing that bacterium. This study aimed for isolation of endolysin from E. coli bacteriophages, and administering them systemic in vivo lab animal and measure the therapeutic efficacy, as well as evaluation of their biosafety for treatment of multi-drugs resistant Escherichia coli in urinary tract infection. This study was performed from September 2019 to February 2020, up to 30 bacteriological samples of uropathogenic Escherichia coli were collected, and examined with their anti-biogram, then bacteriophage cocktails were prepared for three resistant strains of them. Endolysins were extracted from their corresponding bacteriophages, they were characterized; the enzymatic and antibacterial activities of extracted endolysins were tested by in vitro assays; in addition, in vivo experiment was carried out on rabbit animals regarding the therapeutic effect of these extracted endolysins on UTI-causing E. coli. This study showed that the extracted endolysin from highly lytic coliphages were successful in reducing bacterial count in urine of treated rabbit animals which received specific endolysin via venous catheter. Endolysin can be extracted directly from their bacteriophages and being purified and used. The injection of rabbits with UTI with the proper dose of the extracted endolysin was effective in eradicating UTI in all of the treated animals.