Introduction Weaning as a crucial phase for the health of farm monogastric animals, which often involves the use of antibiotics. To limit such use and improve gut health, several nutritional solutions were evaluated. The impact of these solutions was studied both in vitro, focusing on the growth rate of Escherichia coli and Enterococcus faecalis, and in vivo, by examining the intestinal microbiota of weaned piglets. Materials and methods After an in vitro gastro-intestinal digestion (at 37°C – shaking - 4H), 2 strains, E. coli (CIP 54.127) and E. faecalis (CIP 76.117), were incubated in anae-aerobic conditions with the 3 ingredients tested: Ca-butyrate, chestnut tannins and modified dextrin; their respective combinations and a control digestate (ultra-pure water). Exposition concentration ranged between 3 and 10 mg/ml for the combination and the control digestate; the concentration of other ingredients alone and two by two mixes depended on the proportion tested. A total of 192 weaned piglets were divided into six groups receiving the different nutritional solutions and one control group. Piglets’ weight and mortality rate were recorded all along the study as well as the individual antibiotic treatments, if any. A total of 694 fecal swabs were collected by sampling 2 pigs per pen at arrival, on day 14 and day 31 for subsequent 16S V3-V4 targeted metagenomic analysis. Results For both strains, chestnut tannins reduced the growth rate compared to the control digestate (NS). Significant E. coli growth rate reduction (p-value < 0.05) was observed with Ca-butyrate combined with modified dextrin and all 3 ingredients combined. At the end of the in vivo study, piglets of the control group showed the highest mortality rate (~15%) compared to the treated groups while those receiving Ca-butyrate presented no mortality. Fecal microbiota analysis showed no dramatic changes in the bacterial community between groups. However, significant overrepresentation of ASVs belonging to the Christensenellaceae R7 group was observed with the modified dextrin with and without chestnut tannins and all 3 ingredients combined. Conclusion The nutritional solution tested have different impact on gut bacteria with potential to improve weaned piglets’ health and gut bacterial composition balance.