The market for agricultural biologicals as replacements for synthetic crop chemicals has grown significantly due to their ability to improve soil health while reducing the overall carbon footprint. In particular, bio stimulants can enhance crop yield and quality, increase soil microbial activity, and provide other various benefits to support crop productivity. Additionally, a decline in crude protein content in crops—caused by plant breeding, climate change, declining soil organic matter, and changes in environmental legislation— has led to an increased demand for strategies to boost protein levels in staple cereal crops. Ergothioneine (EGT), an amino acid with recognized nutraceutical and micronutrient properties, has gained popularity for its anti-inflammatory and antimicrobial properties on human health. EGT has been studied in relation to cardiometabolic diseases, anti-aging, neurodegenerative disorders, and various other inflammatory conditions. While plants and humans cannot biosynthesize EGT, its production by Streptomyces coelicolor presents as a promising bio-stimulant to support overall plant health and human health. Our study investigates the potential for Streptomyces coelicolor M145 to enhance EGT levels in spring wheat (Triticum aestivum). In vitro analyses quantified intracellular EGT production, under nutrient-rich and minimal nutrient conditions, using a developed cell extract protocol to determine how intracellular EGT levels vary based on condition and time. Following inoculation of S. coelicolor on T. aestivum roots, EGT was extracted and quantified from plant roots and shoots. Results confirmed successful EGT extraction from bacterial cell extracts and plant tissues. Additionally, a fluorescent confocal microscopy staining and imaging protocol was developed to assess bacterial colonization on T. aestivum and its potential as a root endophyte. As soon as day 5 post-inoculation, microscopy revealed clear colonization of S. coelicolor inhabiting T. aestivum roots, shoots, and internodes.