It has been proposed that exposure to infections and microbes protects against atopic diseases, but epidemiological data has so far been conflicting. We hypothesized that maternal exposure to infections and microbes before or during pregnancy would be of particular importance. To test this hypothesis, we studied the incidence of wheezing and atopic dermatitis (AD) in infants of mothers employed in child-care institutions – and thus presumably being highly exposed to infections and microbes – compared with infants of mothers not so employed. A total of 31471 mother-child pairs enroled in the Danish National Birth Cohort were followed prospectively. Information on wheezing episodes, AD, maternal employment, and other variables were collected by interview at 12 and 30 wk of gestation, and 6 and 18 months of age, and by linkage to the Danish Medical Birth Register and the Child-care Database. The relative risk was estimated in Cox proportional hazard models. Analyses were stratified by sibling status (first born or not), as older siblings are likely to be a significant source of infectious agents. The adjusted relative risks of wheeze, recurrent wheeze and AD was 1.14 (95% CI: 0.96–1.37), 1.37 (95% CI: 1.05–1.77), and 1.03 (95% CI: 0.81–1.31), respectively, for first-born infants of mothers employed in child-care institutions compared with infants of mothers not so employed. There was no effect of maternal employment in child-care institutions among infants with older siblings. In conclusion, the results did not support the hypothesis that maternal microbial exposure before or during pregnancy as reflected by maternal employment in child-care institutions protects the offspring against infant wheeze and AD.