Early intervention and short-duration treatments with antibiotics in premature infants are reported to reduce the incidence of necrotizing enterocolitis (NEC), a terrible disease with severe inflammation and impaired intestinal barrier properties. Yet, it is unclear how antibiotics exposure, as well as route of administration used for dosing, can minimize the risk of NEC. With this study, we aimed to investigate if and how administration of antibiotics may affect the barrier properties of intestinal mucosa and mucus. We compared how parenteral (PAR) and a combination of enteral and parenteral (ENT+PAR) ampicillin and gentamicin given to preterm born piglets within 48 h after birth affected both barrier and physical properties of ex vivo small intestinal mucosa and mucus. Permeation of the markers mannitol, metoprolol, and fluorescein-isothiocyanate dextran of 4 kDa (FD4) and 70 kDa (FD70) through the mucosa and mucus was evaluated. For all markers, permeation through the mucosa and mucus collected from PAR piglets tended to be reduced when compared to that observed using untreated piglets. In contrast, permeation through the mucosa and mucus collected from ENT+PAR piglets tended to be similar to that observed for untreated piglets. Additionally, rheological measurements on the mucus from PAR piglets and ENT+PAR piglets displayed a decreased Gʹ and Gʹ/Gʺ ratio and decreased viscosity at 0.4 s⁻¹ as well as lower stress stability compared to the mucus from untreated piglets. Graphical Abstract: [Figure not available: see fulltext.].