Phage endolysins have received increasing attention as potent antibacterial agents. However, although safety evaluation is a prerequisite for the drug development process, a good laboratory practice (GLP)-compliant safety evaluation has not been reported for phage endolysins. A safety evaluation of intravenously administered SAL200 (containing phage endolysin SAL-1) was conducted according to GLP standards. No animals died in any of the safety evaluation studies. In general toxicity studies, intravenously administered SAL200 showed no sign of toxicity in rodent single- and repeated-dose toxicity studies. In the dog repeated-dose toxicity test, there were no abnormal findings, with the exception of transient abnormal clinical signs that were observed in some dogs when daily injection of SAL200 was continued for more than 1 week. In safety pharmacology studies, there were also no signs of toxicity in the central nervous and respiratory system function tests. In the cardiovascular function test, there were no abnormal findings in all tested dogs after the first and second administrations, but transient abnormalities were observed after the third and fourth administrations (2 or 3 weeks after the initial administration). All abnormal findings observed in these safety evaluation studies were slight to mild, were apparent only transiently after injection, and resolved quickly. The safety evaluation results for SAL200 support the implementation of an exploratory phase I clinical trial and underscore the potential of SAL200 as a new drug. We have designed an appropriate phase I clinical trial based on the results of this study.