Urinary tract infections (UTIs) are mainly caused by uropathogenic Escherichia coli (UPEC), whose impact can be exacerbated by multidrug-resistant (MDR) strains. Effective control strategies are, therefore, urgently needed. Among them, phage therapy represents a suitable alternative. Here, we describe the isolation and characterization of novel phages from wastewater samples, as well as their lytic activity against biofilm and adherence of UPEC to HEp-2 cells. The results demonstrated that phage vB_EcoM-phiEc1 (phi Ec1) belongs to Myoviridae family, whereas vB_EcoS-phiEc3 (phi Ec3) and vB_EcoS-phiEc4 (phi Ec4) belong to Siphoviridae family. Phages showed lytic activity against UPEC and gut commensal strains. Phage phi Ec1 lysed UPEC serogroups, whereas phages phi Ec3 and phi Ec4 lysed only UTI strains with higher prevalence toward the O25 serogroup. Moreover, phages phi Ec1 and phi Ec3 decreased both biofilm formation and adherence, whereas phi Ec4 was able to decrease adherence but not biofilm formation. In conclusion, these novel phages showed the ability to decrease biofilm and bacterial adherence, making them promising candidates for effective adjuvant treatment against UTIs caused by MDR UPEC strains.