<p>The objective of this work was to obtain postbiotics derived from the cell wall of <i>Saccharomyces cerevisiae</i> RC016 by applying different strategies and to characterize them in terms of their antitoxin capacity in vitro and their in vivo impact on intestinal integrity, evaluating the modulation of the microbiota and the histomorphometry of the intestine. Moreover, the impact of dried strategies on chemical groups related to food toxin adsorption was analyzed. Nine mechanical and enzymatic cell disruption treatments were assayed using <i>S. cerevisiae</i> RC016 biomass to obtain the postbiotic under study. Then, postbiotics were characterized using high-resolution optical microscopy and assayed for in vitro studies related to their antitoxin activity (adsorption and degradation of aflatoxin B₁). Postbiotics were dried using freeze-dried and spray-dried methods and subjected to FT-IR spectroscopy. Finally, the postbiotic efficacy was determined on an in vivo study conducted on 16 male and female BALB/c mice, divided into two experimental groups, each experimental group (<i>n</i> = 8) separated by sex in different cages (four females and four males): untreated (control) and yeast wall treated (YW); (a) female control; (b) male control; (c) female control + YW; (d) male control + YW. The intestinal microbiota showed significant differences in the counts of LAB and enterobacteria between male and female animals. The histomorphometric analysis showed a significant increase in villi height and width, as well as crypt depth, compared to the control group in male mice with the addition of the postbiotic solution of <i>S. cerevisiae</i>. These findings open new avenues for further optimizing postbiotics’ production processes and evaluating their efficacy across diverse conditions and populations.</p>