Asthma is common in children with allergens and mould influencing the development of the disease. Since children spend most of their time outside their homes within the school environment, school indoor air quality can directly influence their respiratory health. This study aims to identify microbiological contaminants in Maltese schools. The association between contaminants, respiratory health and school characteristics has been analysed. Five primary schools were selected with 9 to 11 years old students participating. Standardised health questionnaires, lung function tests, and school characterisation were performed. Dust samples were analysed for fungi, bacteria and allergens were performed. Penicillium/Aspergillus/Paecilomyces/Variotii (PenAsp) group had the highest median indoor concentration followed by Mycobacterial and Streptomyces species. There was a significant negative correlation between PenAsp and Mycobacterium spp levels in all the participating schools (r = −0.42; p = 0.03). Cat allergen in classroom dust correlated positively with the number of cat owners (r = 0.43; p = 0.041). High exposure to fungi, bacteria and allergens was significantly associated with upper and lower airway atopy. School/classroom characteristics and cleaning protocols were significantly associated with exposure to these pollutants. In conclusion, fungi, bacteria, endotoxin, cat and dog allergens have been found to have a direct influence on school indoor air quality in the Maltese Islands. A significant association was observed between these contaminants and upper and lower airway atopy. Specific school, classroom, cleaning and maintenance characteristics have been identified as having a direct impact on indoor air quality.