Abstract Background Gut bacteria-derived short-chain fatty acids (SCFA) and branched-chain fatty acids (BCFA) are considered to have beneficial metabolic, anti-inflammatory as well as anti-carcinogenic effects. Previous preclinical studies indicated bidirectional interactions between gut bacteria and the chemotherapeutic capecitabine or its metabolite 5-FU. This study investigated the effect of three cycles of capecitabine on fecal SCFA and BCFA levels and their associations with tumor response, nutritional status, physical performance, chemotherapy-induced toxicity, systemic inflammation and bacterial abundances in patients with colorectal cancer (CRC). Methods Forty-four patients with metastatic or unresectable CRC, scheduled for treatment with capecitabine (± bevacizumab), were prospectively enrolled. Patients collected a fecal sample and completed a questionnaire before (T1), during (T2) and after (T3) three cycles of capecitabine. Tumor response (CT/MRI scans), nutritional status (MUST score), physical performance (Karnofsky Performance Score) and chemotherapy-induced toxicity (CTCAE) were recorded. Additional data on clinical characteristics, treatment regimen, medical history and blood inflammatory parameters were collected. Fecal SCFA and BCFA concentrations were determined by gas chromatography–mass spectrometry (GC–MS). Gut microbiota composition was assessed using 16S rRNA amplicon sequencing. Results Fecal levels of the SCFA valerate and caproate decreased significantly during three cycles of capecitabine. Furthermore, baseline levels of the BCFA iso-butyrate were associated with tumor response. Nutritional status, physical performance and chemotherapy-induced toxicity were not significantly associated with SCFA or BCFA. Baseline SCFA correlated positively with blood neutrophil counts. At all time points, we identified associations between SCFA and BCFA and the relative abundance of bacterial taxa on family level. Conclusions The present study provided first indications for a potential role of SCFA and BCFA during capecitabine treatment as well as implications for further research. Trial registration The current study was registered in the Dutch Trial Register (NTR6957) on 17/01/2018 and can be consulted via the International Clinical Trial Registry Platform (ICTRP).