Measurement of biological particles (bioaerosols) in ambient air is of great importance as it is directly linked with the health issues. However, data on the bioaerosols characterization are scarce. Here we report on the concentration and characterization of bioaerosols (including bacterial and fungal aerosols) as well as determination of organic and elemental carbon (OC and EC) in total suspended particulate matter (TSPM) at a busy roadside restaurants-cluster site in New Delhi. In addition, 14 carbonyl volatile organic compounds (carbonyl VOCs) were also measured and their relationship with bioaerosols and OC/EC is assessed. The culturable airborne bacterial and fungal concentrations (CAB and CAF) at restaurant area varied significantly in different seasons ranging from 1.7 × 104–9.8 × 104 (averaged 6.3 × 104 ± 2.6 × 104 cfu m–3) and 3.5 × 102–9.5 × 103 (3.9 × 103 ± 3.1 × 103 cfu m–3) cfu m–3, respectively. Major concentration peaks of TSPM, OC, EC as well as bacterial and fungal aerosols were found in winter and spring seasons. These peaks can be attributed to the low atmospheric boundary layer (ABL) height and favourable meteorological conditions for microbial growth in winter and spring seasons in New Delhi. Good correlations (R2 > 0.5) were observed between CAB, CAF, TSPM and OC. On the other hand, CAB and CAF were not found to be correlated with carbonyl compounds (R2 < 0.2) indicative of their diverse sources. The bacterial identification was done by 16s rDNA sequencing and the identified strains were Bacillus sp., Bacillus firmus, Bacillus licheniformis, Bacillus cereus, Bacillus pumilus, Acinetobacter sp. and Acinetobactor radioresistens gene. Predominant fungal genera identified were Aspergillus, Cladosporium, Alternaria and Fusarium, which are known for adverse health effects causing numerous allergic and pathogenic inflammations.