This study investigates the antimicrobial activity of lipids extracted from Hermetia illucens (black soldier fly) larvae reared on different substrates. The research aims to understand how the fatty acid (FA) composition of the larvae's lipid profile is influenced by the feeding substrate and how this affects the antimicrobial properties of the lipids. The study evaluated the antimicrobial activity of lipids extracted from larvae fed on strawberry, tangerine, and fresh dairy manure substrates against Gram-positive bacteria Micrococcus flavus and Gram-negative bacteria Escherichia coli. The results showed that lipids extracted from larvae reared on strawberry, tangerine, and fresh dairy manure substrates exhibited significant antimicrobial activity against M. flavus, with growth inhibition zones ranging from 1.38 to 1.51 mm. Only the larvae reared on manure showed antimicrobial activity against E. coli. The fatty acid profiles of the larvae were analyzed, and significant differences were found in the levels of C4:0, C10:0, C16:1, C18:3 n3 (ALA), and C20:1. However, the fatty acid profile of the larvae was not significantly influenced by the substrate fatty acid profile, except for C18:0 and C18:2 CIS n6 (LA). The study highlights the potential of black soldier fly larvae as a sustainable source of antimicrobial compounds, particularly their lipids, which can be used in various applications such as feed, cosmetics, and biodiesel. The findings suggest that the antimicrobial activity of the lipids is influenced by factors such as the rearing conditions, larval development stages, and other nutrients like carbohydrates. The research contributes to the understanding of the antimicrobial properties of lipids extracted from black soldier fly larvae and their potential as future therapeutics.This study investigates the antimicrobial activity of lipids extracted from Hermetia illucens (black soldier fly) larvae reared on different substrates. The research aims to understand how the fatty acid (FA) composition of the larvae's lipid profile is influenced by the feeding substrate and how this affects the antimicrobial properties of the lipids. The study evaluated the antimicrobial activity of lipids extracted from larvae fed on strawberry, tangerine, and fresh dairy manure substrates against Gram-positive bacteria Micrococcus flavus and Gram-negative bacteria Escherichia coli. The results showed that lipids extracted from larvae reared on strawberry, tangerine, and fresh dairy manure substrates exhibited significant antimicrobial activity against M. flavus, with growth inhibition zones ranging from 1.38 to 1.51 mm. Only the larvae reared on manure showed antimicrobial activity against E. coli. The fatty acid profiles of the larvae were analyzed, and significant differences were found in the levels of C4:0, C10:0, C16:1, C18:3 n3 (ALA), and C20:1. However, the fatty acid profile of the larvae was not significantly influenced by the substrate fatty acid profile, except for C18:0 and C18:2 CIS n6 (LA). The study highlights the potential of black soldier fly larvae as a sustainable source of antimicrobial compounds, particularly their lipids, which can be used in various applications such as feed, cosmetics, and biodiesel. The findings suggest that the antimicrobial activity of the lipids is influenced by factors such as the rearing conditions, larval development stages, and other nutrients like carbohydrates. The research contributes to the understanding of the antimicrobial properties of lipids extracted from black soldier fly larvae and their potential as future therapeutics.