Simple SummaryThe use of insect lipids as an alternative ingredient is an emergent topic in animal nutrition due to their antimicrobial activities. The present study evaluated the in vitro antimicrobial activities of two insect fats (black soldier fly (Hermetia illucens (HI) fat and yellow mealworm (Tenebrio molitor (TM) fat) and their effect as a total substitute for dietary soybean oil in cecal fermentation and gut microbiota of growing rabbits. The obtained results showed the potential of HI and TM fats as an antibacterial feed ingredient with a positive influence on the rabbit cecal microbiota. HI and TM fats therefore may be a sustainable lipid alternative to soybean oil in rabbit nutrition with possible interesting applications in the feed industry.This study aimed to evaluate the in vitro antimicrobial activities of two types of insect fats extracted from black soldier fly larvae (HI, Hermetia illucens L.) and yellow mealworm larvae (TM, Tenebrio molitor L.) and their effects as dietary replacement of soybean oil (S) on cecal fermentation pattern, and fecal and cecal microbiota in rabbits. A total of 120 weaned rabbits were randomly allotted to three dietary treatments (40 rabbits/group) —a control diet (C diet) containing 1.5% of S and two experimental diets (HI diet (HID) and TM diet (TMD)), where S was totally substituted by HI or TM fats during the whole trial that lasted 41 days. Regarding the in vitro antimicrobial activities, HI and TM fats did not show any effects on Salmonella growth. Yersinia enterocolitica showed significantly lower growth when challenged with HI fats than the controls. The insect fat supplementation in rabbit diets increased the contents of the cecal volatile fatty acids when compared to the control group. A metataxonomic approach was adopted to investigate the shift in the microbial composition as a function of the dietary insect fat supplementation. The microbiota did not show a clear separation as a function of the inclusion, even if a specific microbial signature was observed. Indeed, HI and TM fat supplementation enriched the presence of Akkermansia that was found to be correlated with NH3-N concentration. An increase in Ruminococcus, which can improve the immune response of the host, was also observed. This study confirms the potential of HI and TM fats as antibacterial feed ingredients with a positive influence on the rabbit cecal microbiota, thus supporting the possibility of including HI and TM fats in rabbit diets.