Oral administration of omega-3 polyunsaturated fatty acids (PUFAs) to rodents and humans is associated with an increase in gut bacteria that are predicted to synthesise short-chain fatty acids (SCFAs). We tested the hypothesis that physiological levels of omega-3 PUFAs in the distal intestinal lumen (1–50 μg/mL) are associated with increased SCFA synthesis in an in vitro fermentation model using faecal slurry from 10 healthy participants (mean age 30 years), with and without exogenous dietary fibres. SCFAs were measured by gas chromatography-flame ionisation detection (n = 10), and changes in bacterial composition were analysed by shotgun metagenomic sequencing (n = 6). In the presence of omega-3 PUFAs, there was a mean 9.3% (no inulin; P = 0.03) and 19.3% (+ 0.01 mg/mL inulin; P = 0.01) increase in total SCFA concentration at 24 h compared with paired control fermentations. Omega-3 PUFAs had a limited effect on the fermentation model microbiome in the absence of inulin. However, omega-3 PUFAs (50 μg/mL) were associated with increased abundance of Bifidobacteriaceae compared with paired control fermentations, if inulin (0.01 mg/mL) was present. Prebiotic activity of omega-3 PUFAs drives SCFA synthesis in an in vitro colonic fermentation model and is augmented by the soluble fibre inulin.