The pre- and postnatal environment may represent a window of opportunity for allergy and asthma prevention, and the hygiene hypothesis implies that microbial agents may play an important role in this regard. Using the cowshed-derived bacterium Acinetobacter lwoffii F78 together with a mouse model of experimental allergic airway inflammation, this study investigated the hygiene hypothesis, maternal (prenatal) microbial exposure, and the involvement of Toll-like receptor (TLR) signaling in prenatal protection from asthma. Maternal intranasal exposure to A. lwoffii F78 protected against the development of experimental asthma in the progeny. Maternally, A. lwoffii F78 exposure resulted in a transient increase in lung and serum proinflammatory cytokine production and up-regulation of lung TLR messenger RNA. Conversely, suppression of TLRs was observed in placental tissue. To investigate further, the functional relevance of maternal TLR signaling was tested in TLR2/3/4/7/9(-/-) knockout mice. The asthma-preventive effect was completely abolished in heterozygous offspring from A. lwoffii F78-treated TLR2/3/4/7/9(-/-) homozygous mother mice. Furthermore, the mild local and systemic inflammatory response was also absent in these A. lwoffii F78-exposed mothers. These data establish a direct relationship between maternal bacterial exposures, functional maternal TLR signaling, and asthma protection in the progeny.