Allergies became a major public health problem, identified as an important global pandemic with a considerable impact on the worldwide economy. In addition, a higher prevalence of pollen Type I sensitization cases in urban environments in comparison with the rural territories was detected. Our survey sought to assess the main biological pollution episodes caused by the aeroallergens of the major allergenic tree species in urban environments. A Hirst-type volumetric device was used for pollen sampling and a Burkard Cyclone sampler for the detection of tree atmospheric allergens over two years. The main allergens of Alnus, Fraxinus, Betula, Platanus and Olea, were detected in the atmosphere. Three peaks of important pollen concentrations were recorded throughout the year. The developed regression equations between pollen counts and allergen proteins registered great R2 values. The number of days with probability of allergenic symptoms was higher when the pollen and allergen data were assessed altogether. Fraxinus allergens in the atmosphere were detected using Ole e 1 antibodies and the Aln g 1 allergens with Bet v 1 antibodies, demonstrating the cross-reaction processes between the principal allergenic proteins of the Oleaceae and Betulaceae families. Long Distance Transport processes (LDT) showed that pollen from Betula populations located in mountainous areas increased the secondary peaks of pollen and allergen concentrations, and air masses from extensive olive orchards of North-Eastern Portugal triggered the highest concentrations in the atmosphere of Olea pollen and Ole e 1 allergens.