The seed microbiota has great potential for enhancing agricultural practices and promoting sustainable plant growth. However, excessive and improper application of agrochemicals may have unforeseen effects on the seed microbiota, posing potential risks to crop health and even ecological integrity. Despite this, the impact of agrochemicals on seed microbiota in domesticated crops remains largely unexplored. Here, we aimed to analyze the effects of four common agrochemicals on the seed microbiome of highland barley in Tibet using high-throughput sequencing. Community diversity analysis suggests that the differences in seed microbial groups under different agrochemical treatments are not significant. Interestingly, we found that the key microbial taxa in barley seeds responded distinctly to specific agrochemicals. Lactobacillus spp. were identified as positive biomarkers in response to tebuconazole and lambda-cyhalothrin due to an increase in relative abundance as a probiotic. Conversely, Leuconostoc and Helicobacter were recognized as positive biomarkers upon exposure to pydiflumetofen, attributed to a notable reduction in the abundance of pathogens. Collectively, our findings have revealed the unique responsiveness of seed microbial taxa to agrochemical exposure. Further mechanistic insights into the responsiveness of these microbial biomarkers to agrochemicals may establish a foundational framework for microbiome-targeted verification of agrochemical application, ensuring food safety throughout the cultivation of Tibetan barley.