Abstract: To screen for endophytic actinomycetes with antagonistic properties against Phytophthora nicotiana and growth-promoting characteristics, this study employed tissue separation methods to isolate and purify endophytic actinomycetes from healthy tobacco plants sourced from the Sanmenxia region. Additionally, the isolation frequencies of these strains were analyzed across different plant tissues and growth stages. Initial screening was conducted using confrontation culture methods, followed by rescreening via mycelial growth rate assays to evaluate the antagonistic activity of the endophytic actinomycetes against P. nicotiana. The ability of the strains to produce 3-indoleacetic acid (IAA) was determined using the Salkowski colorimetric assay. The taxonomic status of the antagonistic and growth-promoting strains was determined through morphological, physiological, biochemical, and molecular biological characterization. A total of 250 endophytic actinomycete strains were isolated. The isolation frequencies across different plant parts were as follows: root > leaf > stem, at 68.80%, 19.20%, and 12.00%, respectively. Across different growth stages, the frequencies were: rosette growth stage > prosperous growth stage > maturation stage, at 47.20%, 42.40%, and 10.40%, respectively. Initial screening identified 64 strains with antagonistic activity exceeding 60.00% against P. nicotiana, and rescreening yielded 8 strains with inhibition rates ranging from 16.06% to 70.89%. Seven strains were found to produce IAA, with yields ranging from 17.15 to 40.09 μg/mL. Based on the combined results of initial and rescreening inhibition rates and IAA production, strains T-66 and C-77 were selected as candidate strains and were identified as Streptomyces pratensis and S. rochei, respectively. These two strains identified in this study provide new microbial resources for the biological control of tobacco black shank disease.