Background: The role of mite allergen exposure in sensitization and development of asthma has been widely recognized. Previous studies have shown that Dermatophagoides pteronyssinus and Blomia tropicalis were the most prevalent house dust mites in Brazil, while D. farinae was rarely found. The aim of this study was to measure Der f 1 and Der p 1 allergen levels in Brazilian asthmatics’ and controls’ homes. Methods: Sixty-four houses (32 asthmatic, 32 control) were visited for dust sampling from five sites. Der f 1 and Der p 1 levels were measured by two-site monoclonal-antibody-based ELISAs. Results: The highest levels of Der f 1 and Der p 1 allergens were found in bedding samples from both asthmatics’ and controls’ homes. However, the geometric mean of Der f 1 levels (15.8 μg/g of dust) was significantly higher than for Der p 1 (8.2 μg/g of dust) in these samples. In addition, allergen levels ≥10 μg/g of dust were found in 60–80% of the samples for Der f 1 and about 50% for Der p 1. Conclusions: These results indicate that high levels of Der f 1 allergen are present in both asthmatics’ and controls’ homes, in contrast to previously reported data. Therefore, studies on exposure to mites should be performed in different cities, seasons and times, since the mite fauna might be subject to variations. Knowledge of the mite fauna will certainly improve the means of investigating the association between allergen exposure and sensitization, allowing to establish the inclusion of new mite extracts in inhalant skin test sets, and even to detect monosensitized patients with respiratory allergy.