Due to the increase in the number of cases of infections by multidrug-resistant microorganisms, it became necessary to advance in new therapeutic alternatives, such as phage therapy, to contain the spread of these microorganisms. Among the pathogens that have the most alarming mechanisms virulence and resistance, a group called ESKAPE contains six bacteria that are considered to be the leading cause of healthcare-associated infections worldwide (Enterococcus faecium; Staphylococcus aureus; Klebsiella pneumoniae; Acinetobacter baumannii; Pseudomonas aeruginosa; Enterobacter spp) and are classified as serious threats, due to the wide dissemination of antimicrobial resistance genes, which leads to increased mortality associated with these infections. The use of bacteriophages to treat bacterial infections (Phage therapy) is gaining visibility in Western countries, which has recently led the FDA to allow this practice to be conducted in the USA. Therefore, this study aimed to isolate and characterize six different bacteriophages isolated from hospital sewage, called vB_EfaM-Ef1000, vB_SaP-Sa491, vB_KpS-Kp3978, vB_AbM-Ab1219, vB_PaS-Pa1461 and vB_EcS-Ec4815, with action against ESKAPE bacteria. Transmission electron microscopy has shown that isolated phages are classified within three families (Myoviridae, Siphoviridae and Podoviridae) of the order Caudovirales, as they have different sizes and shapes of tails. Phages were submitted to biophysical stability tests, demonstrating viability in low and high pH ranges (3.5 to 11.5) and different temperatures (-20, 4, 25 and 30 °C). Different multiplicities of infection (MOI) were tested in the bacterial challenge test, an assay performed in a 96-well plate to document phage virulence. vB_SaP-Sa491 and vB_KpS-Kp3978 were able to inhibit bacterial growth for 18 hours, which showed a significant difference of up to 4 logs (CFU/mL) compared to the control, proven by the recovery of the bacteria after the test. In addition, clinical samples of multidrug-resistant ESKAPE isolated from tissue, tracheal secretions and urine were sensitive to phage in host range assays, documented by the formation of zones of inhibition (plaques) in agar cultures. The possibility of combining these bacteriophages in cocktails, or associating them with antimicrobials to fight infections in the ESKAPE group represents a therapeutic strategy with great potential, being an alternative for reducing morbidity and mortality from healthcare-associated infections.