The variation in surface characteristics and the composition of extracellular polymeric substances (EPS) of bacterial cells during biodegradation of single and multi-aromatic hydrocarbons was investigated in the present study. The maximum cell surface hydrophobicity (CSH) of 80.1% was observed during the degradation of toluene. Bacterial cells acquired more negative surface charge with an increase in CSH and vice versa. Proteins constituted the major fraction of EPS during biodegradation of benzene and toluene with protein/carbohydrate ratio varying between 2.19 and 3.1. Carbohydrates constituted the major fraction of EPS in the presence of pyridine. A significant variation in cell surface characteristics was observed in multi-substrate systems involving heterocyclic and monocyclic aromatic hydrocarbons. An increase in EPS production (62.89 mg/g) did not facilitate enhanced degradation of hydrophobic substrates in multi-substrate system involving benzothiophene, benzofuran, benzene and toluene. Under toxic conditions, especially at higher concentration of target pollutants, a significant increase in concentration of polysaccharides was observed compared to proteins. © 2017, © 2017 Informa UK Limited, trading as Taylor & Francis Group.