The effect of several types of whey milk - cow, sheep, goat and a mixture of them (60 : 20 : 20, respectively) - was assessed in the human gut microbiota. The prebiotic potential of these substrates was evaluated through in vitro gastrointestinal digestion following faecal batch culture fermentations (mimicking colonic fermentation) for 48 hours, using faeces from normal-weight (NW) and obese (OB) donors. Throughout the fermentation process, pH, gas production, short chain and branched fatty acids (SCFA-BCFA) were measured, as well as the changes of microbiota using qPCR. The pH decreased in all whey samples during the fermentation process. Gas production was higher in all whey samples than in controls, especially at 12 hours (p < 0.05). The diversity of SCFA and BCFA production was significantly different between the donors, in particular cow and mixed whey. Whey milk had a strong prebiotic effect on the gut microbiota of NW and OB donors, showing a significant increase of Bifidobacterium (p < 0.05) with cow, sheep and mixed whey and increase in the Lactobacillus group, particularly in OB donors. Bacteria associated with obesity did not show an increase in any of the groups of donors. Therefore, supplementing a diet with these types of whey can selectively stimulate the growth of probiotic bacteria, enhancing SCFA production, which could improve intestinal disorders. In addition, it may be an interesting approach to the prevention of overweight and obesity and related diseases. Whey milk has a potent prebiotic effect. It can selectively stimulate desirable bacteria and SCFA profile, in both OB and NW donors, contributing to improved intestinal health and reducing obesity.