Abstract: The actinomycete Streptomyces lydicus A01 promotes tomato seedling growth; however, the underlying mechanism is unclear. In this study, we investigated whether changes in soil microbial diversity, following Streptomyces lydicus A01 treatment, were responsible for the increased tomato seedling growth. Eukaryotic 18S ribosomal DNA (rDNA) sequencing showed that S. lydicus A01-treated and untreated soil shared 193 operational taxonomic units (OTUs), whereas bacterial 16S rDNA sequencing identified 1,219 shared OTUs between the treated and untreated soil. Of the 42 dominant eukaryotic OTUs, eight were significantly increased and six were significantly decreased after A01 treatment. Of the 25 dominant bacterial OTUs, 12 were significantly increased and eight were significantly decreased after A01 treatment. Most of the eukaryotes and bacteria that increased in abundance exhibited growth promoting characteristics, which were mainly predicted to be associated with mineralization of nitrogen and phosphorus, phosphate solubilization, nutrient accumulation, and secretion of auxin, whereas some were related to plant protection, such as the degradation of toxic and hazardous substances. Soil composition tests showed that S. lydicus A01 treatment enhanced the utilization of nitrogen, phosphorus, and potassium in tomato seedlings. Thus, microbial fertilizers based on S. lydicus A01 may improve plant growth, without the detriment effects of chemical fertilizers.