Developing and applying a novel plant growth–promoting agent (PGPA; a micronutrient-amino acid chelated compound developed from autolysis yeast cells) in alleviating salt stress toxicity can be the best alternative option environmentally and economically. High-performance liquid chromatography (HPLC) showed that the assembled PGPA is rich in nucleobases than yeast extract (> 56-fold). This study, as a first investigation, was aimed to evaluate PGPA’s potential role in reducing oxidative injury induced by salt stress. Barley (Hordeum vulgare L. cv. Giza 123) plants were grown under non-saline or saline conditions (6.0 and 12.0 dS m−1) with and without PGPA foliar application. The PGPA application mitigated salt-induced oxidative stress by enhancing the activity of superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, glutathione peroxidase, and glutathione S-transferase, as well as the content of ascorbate, glutathione, proline, and glycinebetaine. Moreover, PGPA protected salt-stressed plants from the deleterious effects of methylglyoxal by up-regulating the glyoxalase enzymes activity. The PGPA alleviated membrane damage by decreasing reactive oxygen species (ROS) accumulation, lipid peroxidation, protein oxidation, electrolyte leakage, and NADP+ content. The protection of photosynthesis by PGPA was closely associated with the improved chlorophyll fluorescence parameters, leaf water content, membrane stability index, and NADPH content. The PGPA-treated plants also exhibited higher stomatal conductivity together with improved transpiration and photosynthetic rates under saline conditions. Overall, PGPA regulated the antioxidant machinery, glyoxalase system, and photosynthetic capacity, implying that it plays a critical role in salt stress mitigation. Therefore, it could be a useful agent to alleviate the harmful effects of salinity stress.