Bacteriophages are viruses that parasitic to bacteria. Bacteriophages or phages can cause total damage to bacteria by lysing the host bacteria. The bacteriophage Escherichia coli is a promising biological control agent for eradicating E. coli. E. coli is a microbial indicator of water pollution that determines the quality of contaminated water. E. coli bacteria can cause diarrhea, urinary tract infections, prostate, and other infections of the extra-intestinal system. The purpose of this study was to determine the optimal temperature for phage effectiveness in lysing E. coli. The temperature were at 20˚C, 30˚C, 40˚C, 50˚C, 60˚C, 70˚C, 80˚C, 90˚C in vitro. This study used descriptive quantitative research using experimental methods. The bacteriophages tested were phages isolates FA 3-3A, FA 3-3B, FB 3-3A, FB 3-3B, FC 1-1A, and FC 1-1B specific for E. coli bacteria (IA 3-3, IB 3 - 3, and IC 1-1). The result of the study shown that the optimal temperature of isolate phage FA 3-3A at 40°C, FA 3-3B at 20°C, FB 3-3A at 50°C, FB 3-3B at temperature 30°C, FC 1-1A at 20°C, and FC 1-1B at 40°C. Keywords: Bacteriophage, Escherichia Coli, Lysis, Optimal, and Temperature.