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Among four mycobacteria, <i>Mycobacterium avium</i>, <i>M. intracellulare</i>, <i>M. bovis</i> BCG and <i>Mycobacteroides</i> (<i>My.</i>) <i>abscessus</i>, we established a silkworm infection assay with <i>My. abscessus</i>. When silkworms (fifth-instar larvae, <i>n</i> = 5) were infected through the hemolymph with <i>My. abscessus</i> (7.5 × 10⁷ CFU/larva) and bred at 37 °C, they all died around 40 h after injection. Under the conditions, clarithromycin and amikacin, clinically used antimicrobial agents, exhibited therapeutic effects in a dose-dependent manner. Furthermore, five kinds of microbial compounds, lariatin A, nosiheptide, ohmyungsamycins A and B, quinomycin and steffimycin, screened in an in vitro assay to observe anti-<i>My. abscessus</i> activity from 400 microbial products were evaluated in this silkworm infection assay. Lariatin A and nosiheptide exhibited therapeutic efficacy. The silkworm infection model with <i>My. abscessus</i> is useful to screen for therapeutically effective anti-<i>My. abscessus</i> antibiotics.