Phytophthora infestans, the notorious pathogen of potato late blight, leads to a severe decline in potato yields and even harvest failure. We isolated 201 endophytic isolates from healthy root tissues of potatoes, among which 41 showed strong antagonistic activity against P. infestans. Further, the tolerance to stress and the potential application against potato late blight of these antagonistic isolates were tested. Most of them were extremely tolerant to stresses such as acid–alkali, temperature, UV, salt, and heavy metal stress. However, some antagonistic isolates with excellent stress tolerance might be pathogenic to potatoes. Combining the screening results, a total of 14 endophytes had excellent comprehensive performance in all the tests. In this paper, the endophyte 6-5 was selected among them for the preliminary exploration of the anti-oomycete mechanism. Analysis of the 16S rDNA sequence revealed that 6-5 had a high homology to the corresponding sequence of Bacillus velezensis (99.72%) from the NCBI database. Endophyte 6-5 significantly inhibited the mycelial growth of P. infestans, with an inhibition rate of over 90% in vitro assays, and deformed the hyphal phenotype of P. infestans. In addition, endophyte 6-5 could secrete protease and cellulase, and produce antagonistic substances with high thermal stability, which might be helpful to its antagonistic activity against P. infestans. Furthermore, it was demonstrated that 6-5 had the ability to improve the resistance of potato tubers to late blight. In short, our study described the process of isolating and screening endophytes with antagonistic activity against P. infestans from potato roots, and further explored the potential of biocontrol candidate strain 6-5 in potato late blight control.