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Biosurfactant immunomodulatory activities in mammals, nematodes, and plants have been investigated. However, the immune activation property of biosurfactants in insects has not been reported. Therefore, here, we studied the defense response triggered by lipopeptides (fengycin and iturin A), glycolipids (rhamnolipid), and cyclic polypeptides (bacitracin) in the coleopteran insect, mealworm <i>Tenebrio molitor</i>. The in vitro antimicrobial activities against Gram-positive (<i>Staphylococcus aureus</i>) and Gram-negative (<i>Escherichia coli</i>) bacteria and fungi (<i>Candida albicans</i>) were assessed by mixing these pathogens with the hemolymph of biosurfactant-immune-activated larvae. <i>E. coli</i> growth was remarkably inhibited by this hemolymph. The antimicrobial peptide (AMP) induction results also revealed that all biosurfactants tested induced several AMPs, exclusively in hemocytes. The survivability analysis of <i>T. molitor</i> larvae challenged by <i>E. coli</i> (10⁶ CFU/µL) at 24 h post biosurfactant-immune activation showed that fengycin, iturin A, and rhamnopid significantly increased survivability against <i>E. coli</i>. Biosurfactant-induced <i>TmSpatzles</i> activation was also monitored, and the results showed that <i>TmSpz3</i> and <i>TmSpz-like</i> were upregulated in the hemocytes of iturin A-injected larvae, while <i>TmSpz4</i> and <i>TmSpz6</i> were upregulated in the fat bodies of the fengycin-, iturin A-, and rhamnolipid-injected larvae. Overall, these results suggest that lipopeptide and glycolipid biosurfactants induce the expression of AMPs in <i>T. molitor</i> via the activation of spätzle genes, thereby increasing the survivability of <i>T. molitor</i> against <i>E. coli</i>.