Background: Long-term consumption of a high-fat diet (HFD) causes obesity and is a risk factor for depression. The HFD has a significant impact on the gut microbiota, and dysbiosis of the microbiota is now associated with certain psychiatric disorders such as anxiety and depression. We aimed to investigate whether modulation by antibiotic treatment of the composition of the gut microbiota in diet-induced obese (DIO) C57BL/6J male mice has an impact on depressive-like behaviour. Methods: In this study, we have analysed the effects of a 15 weeks HFD on helplessness assessed in the forced swim test and anhedonia assessed in the sucrose preference test. Two weeks before the start of the behavioural tests, a group of HFD mice were given a combination of two non-absorbable antibiotics, neomycin and polymyxin B. Results: In DIO mice, anhedonia and significant changes in the composition of the gut microbiota at the phyla and family level were observed. On the other hand, there was no significant effect of HFD on the peripheral inflammatory profile. In DIO mice, antibiotic treatment resulted in very pronounced alteration in the composition of the gut microbiota, without any change in anhedonia behaviour. Conclusion: In DIO mice, only four families of bacteria were not affected in their relative abundance by the antibiotic treatment, the Bifidobacteriaceae, Erysipelotrichaceae, Rikenellaceae and Streptococcaceae. This stability concomitant with that of anhedonia suggests that these families may be involved in anhedonia in DIO mice.