Antibiotic tolerance, the ability of a typically susceptible microorganism to survive extended periods of exposure to antibiotics, has a critical role in chronic and recurrent bacterial infections, and facilitates the evolution of antibiotic resistance. However, the physiological factors that contribute to the development of antibiotic tolerance, particularly in vivo, are not fully known. Despite the fact that a high-fat diet (HFD) is implicated in several human diseases, the relationship between HFD and antibiotic efficacy is still poorly understood. Here, we evaluated the efficacy of multiple clinically relevant bactericidal antibiotics in HFD-fed mice infected with methicillin-resistant Staphylococcus aureus (MRSA) or Escherichia coli. We found that HFD-fed mice had higher bacterial burdens and these bacteria displayed lower susceptibility to bactericidal antibiotic treatment compared with mice that were fed a standard diet, while microbiota-depleted standard-diet- or HFD-fed mice showed similar susceptibility. Faecal microbiota transplantation from HFD-fed mice impaired antibiotic activity in mice fed a standard diet, indicating that alteration of the gut microbiota and related metabolites in HFD-fed mice may account for the decreased antibiotic activity. 16S rRNA sequencing and metabolomics analysis of faecal samples revealed decreased microbial diversity and differential metabolite profiles in HFD-fed mice. Notably, the tryptophan metabolite indole-3-acetic acid (IAA) was significantly decreased in HFD-fed mice. Further in vitro studies showed that IAA supplementation inhibited the formation of bacterial persisters and promoted the elimination of persisters in combination with antibiotic treatment, potentially through the activation of bacterial metabolic pathways. In vivo, the combination of IAA and ciprofloxacin increased the survival rate of HFD-fed mice infected with MRSA persisters. Overall, our data reveal that a HFD has an antagonistic effect on antibiotic treatment in a mouse model, and this is associated with the alteration of the gut microbiota and IAA production.