Ambient levels of culturable bioaerosol were measured at four different sites of Delhi, India in six size ranges (> 7.0 µm, 7.0–4.7 µm, 4.7–3.3 µm, 3.3–2.1 µm, 2.1–1.1 µm, < 1.1 µm). The study also accounted the seasonal variation (monsoon, post monsoon, winter and pre-monsoon) of the air microbes. The sampling was carried out for three different fractions of bioaerosols viz. fungi, gram positive and negative bacteria during August 2010 to April 2011 using a six-stage viable cascade impactor sampler. Unlike gram positive and negative bacteria, the concentration of fungal bioaerosol found in different stages at each site seems to follow a typical pattern in all four season. The typical pattern of concentration depicts that majority of the fungal species found in the diameter range of 3.3–2.1 µm, which coincides with the penetration range in the secondary bronchi of the lungs in the human body. This reveals that majority of the immunotoxic and allergic fungi found at this stage are mostly prone to affect the secondary bronchi in human lungs when inhaled. At all four sites maximum fungal concentration (1740.5–3224.7 CFU m–3), gram-positive bacterial concentration (2790.6–9428.3 CFU m–3) and gram-negative bacterial concentration (1990.3–7609 CFU m–3) were found in post monsoon season. In the majority of the sites, minimum concentrations were found in monsoon period which probably may be due to rain wash during the sampling. For all the three bioaerosol fractions no particular relationship pattern was found to exist between their respective concentrations with temperature and relative humidity (RH). However, higher range of variation was observed at higher concentration levels and lower range of variation at low concentration levels for all the three bioaerosol fractions. Most of the fungal bioaerosol identified such as Penicillium sp., Alternaria sp. and Aspergillus sp. are associated with immunotoxic and allergic diseases.