Chlorpyrifos (CPF), a commonly used organophosphate pesticide, poses potential risks to human health, particularly affecting the gut microbiota (GM), intestinal barrier (IB), and blood-brain barrier (BBB). CPF-induced gut dysbiosis compromises the integrity of both the IB and the BBB, leading to increased intestinal permeability, inflammation, and bacterial translocation, all of which may impact neurological health. Although CPF’s effects on the GM are documented, limited research explores how these impacts differ in women, particularly during pregnancy. To address this gap, this study investigates CPF’s effects using three advanced human in vitro models: the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) to mimic the gut environment of women of child-bearing age and pregnant women, a Caco-2 model for the IB, and a BBB model to assess CPF’s effects and the protective role of the prebiotic inulin. Microbiological analyses of SHIME® supernatants, including bacterial culture and quantification of short-chain fatty acids (SCFAs) and CPF metabolites, were conducted to assess gut composition and pesticide degradation. We also examined the effects of CPF-induced dysbiosis on IB and BBB permeability to FITC-Dextran, focusing on bacterial translocation after 4 hours of exposure to CPF-treated SHIME® supernatants. Our results revealed significant intestinal imbalance, marked by an increase in potentially pathogenic bacteria in the GM of both non-pregnant and pregnant women exposed to CPF. This dysbiosis led to a significant shift in SCFAs ratio and increased IB permeability and bacterial translocation across the IB, but not the BBB. Notably, inulin supplementation restored GM balance and prevented bacterial translocation, highlighting its potential as a preventive measure against CPF-induced dysbiosis. This study enhances our understanding of the health risks associated with CPF exposure in women, with implications for maternal and fetal health, and underscores the importance of considering physiological states such as pregnancy in toxicological research.