This study investigates the antimicrobial activity of lipids extracted from *Hermetia illucens* (black soldier fly) larvae reared on different substrates. The larvae were fed on five substrates: Gainesville diet, strawberry, orange, tangerine, and fresh dairy manure. The antimicrobial activity was evaluated against *Micrococcus flavus* (Gram-positive) and *Escherichia coli* (Gram-negative). The results showed that lipids extracted from larvae reared on strawberry, tangerine, and fresh dairy manure exhibited significant antimicrobial activity against *M. flavus*, with inhibition zones ranging from 1.38 to 1.51 mm. Only the manure substrate showed antimicrobial activity against *E. coli*. The fatty acid profiles of the larvae lipids were analyzed, and significant differences were found for C4:0, C10:0, C16:1, C18:3 n3 (ALA), and C20:1. The study also found that the fatty acid profile of *H. illucens* was not significantly influenced by the substrate, except for C18:0 and C18:2 CIS n6 (LA). This suggests that other factors, such as rearing conditions and larval development stages, also affect the fatty acid composition of insects. The findings highlight the potential of *H. illucens* lipids as natural antimicrobial agents and the importance of considering the rearing substrate in their production.This study investigates the antimicrobial activity of lipids extracted from *Hermetia illucens* (black soldier fly) larvae reared on different substrates. The larvae were fed on five substrates: Gainesville diet, strawberry, orange, tangerine, and fresh dairy manure. The antimicrobial activity was evaluated against *Micrococcus flavus* (Gram-positive) and *Escherichia coli* (Gram-negative). The results showed that lipids extracted from larvae reared on strawberry, tangerine, and fresh dairy manure exhibited significant antimicrobial activity against *M. flavus*, with inhibition zones ranging from 1.38 to 1.51 mm. Only the manure substrate showed antimicrobial activity against *E. coli*. The fatty acid profiles of the larvae lipids were analyzed, and significant differences were found for C4:0, C10:0, C16:1, C18:3 n3 (ALA), and C20:1. The study also found that the fatty acid profile of *H. illucens* was not significantly influenced by the substrate, except for C18:0 and C18:2 CIS n6 (LA). This suggests that other factors, such as rearing conditions and larval development stages, also affect the fatty acid composition of insects. The findings highlight the potential of *H. illucens* lipids as natural antimicrobial agents and the importance of considering the rearing substrate in their production.