The seeds of Nigella sativa Linn. (Ranunculaceae), commonly known as Black cumin, are predominantly used as carminative, antispasmodic, and stimulant. The main objective of the present study was to evaluate the effect of N. sativa seed extract on the permeation of co-infused amoxicillin across the gut wall. The methanolic extract of N. sativa improved intestinal permeability of amoxicillin in in-vitro experiments in a dose-dependent manner. Two new glycosides, decanyl nigelloic acid diglucoside [ n-decanyl-3-aldehydic-4-methoxy-5-hydroxy benzoate-5- β-D-glucofuranosyl (2→1)- β-D-glucopyranosyl-(2→1)- β-D-glucopyranoside]] and nige-labdienoyl triglucoside [homo-labd-5, 9(11)-dien-16-onyl- β-D-glucopyranosyl (2→1)- β-D-glucopyranosyl (2→1)- β-D-glucopyrano-side], along with seven known fatty acid glycerides/esters, were isolated from the gut permeation enhancing extract. The structures of these new glycosides were elucidated by detailed spectroscopic analyses.