The increase in the global incidence of atopic disease and asthma during the past few decades has been ascribed to environmental factors, including the reduction in exposure to serious infections. The hypothetical framework to explain the inverse relationship between infections and atopic disease and asthma has been called the "hygiene hypothesis." Animal and experimental models have identified Mycobacteria as important potential candidates in the hygiene hypothesis by demonstrating that exposure to Mycobacteria or mycobacterial proteins led to subsequent reduction in different atopic manifestations. Although there are epidemiological studies in support, they have not always been consistent. In this review we appraise epidemiologic evidence on the inverse relationship between mycobacterial exposure and atopic disease, explore the immunological mechanisms involved and evidence that this effect may be dose-dependent, and discuss the challenges facing the use of Mycobacteria as vaccine for prevention of atopic disease.