Twelve undescribed glucosyloxybenzyl 2-isobutylmalates, bletistrosides A-L were obtained from the pseudobulbs of Bletilla striata under the guidance of HPLC-PDA. Their structures and absolute configurations were determined by the spectroscopic analyses, along with acidic hydrolysis and alkaline hydrolysis experiments. The neuroprotective effects and the antimicrobial activities of selective compounds were evaluated. Bletistroside G exhibited potential neuroprotective effect against glutamate-induced PC12 cell injury, with improving cell viabilities by 11.24% compared to the model group at 10 μM. Bletistrosides A, B, G, and L showed weak antimicrobial activities against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, however, bletistroside G showed obviously selective inhibition against P. aeruginosa.