Zymomonas mobilis has been identified as a promising cellular factory for biofuels due to its efficient, natural production of and tolerance to ethanol. Recent discovery of ethanol-responsive small regulatory RNAs (sRNAs) in Z. mobilis suggested the potential of exploiting these elements for strain engineering. As global controllers of gene expression, sRNAs represent powerful tools for engineering complex phenotypes. In this study, we screen a set of experimentally confirmed sRNAs in Z. mobilis for their impact on ethanol tolerance and identify sRNA candidates that have significant impact on ethanol tolerance. We have conducted multi-omics analyses (including proteomics, transcriptomics, and sRNA-affinity tag purification with high-throughput sequencing) of these sRNA-engineered strains to map gene networks under the influence of their regulation. This work has led to the finding that these sRNAs regulate unique combinations of enzymes along pathways known to be relevant to ethanol tolerance such as oxidative stress response, ATP production, redox balance, translation, transport, and DNA repair. Importantly, this work represents the first analysis and application of de novo sRNA-driven strain engineering in non-model Z. mobilis that is of relevance to biofuel technologies.