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Magnetobiological effects on growth and virulence have been widely reported in <i>Escherichia coli</i> (<i>E. coli</i>). However, published results are quite varied and sometimes conflicting because the underlying mechanism remains unknown. Here, we reported that the application of 250 mT static magnetic field (SMF) significantly reduces the diameter of <i>E. coli</i> colony-forming units (CFUs) but has no impact on the number of CFUs. Transcriptomic analysis revealed that the inhibitory effect of SMF is attributed to differentially expressed genes (DEGs) primarily involved in carbon source utilization. Consistently, the addition of glycolate or glyoxylate to the culture media successfully restores the bacterial phenotype in SMF, and knockout mutants lacking glycolate oxidase are no longer sensitive to SMF. These results suggest that SMF treatment results in a decrease in glycolate oxidase activity. In addition, metabolomic assay showed that long-chain fatty acids (LCFA) accumulate while phosphatidylglycerol and middle-chain fatty acids decrease in the SMF-treated bacteria, suggesting that SMF inhibits LCFA degradation. Based on the published evidence together with ours derived from this study, we propose a model showing that free radicals generated by LCFA degradation are the primary target of SMF action, which triggers the bacterial oxidative stress response and ultimately leads to growth inhibition.