The influence of PAH chemical structure and concentration, added in either single (75 or 300 mg kg−1) or multiple (2 × 75, 2 × 150 or 4 × 75 mg kg−1) applications as single- or multiple-contaminant systems, on the development of PAH biodegradation in a pristine soil was investigated. Development in microbial catabolic ability was assessed at 0, 28, 56 and 84 d by monitoring 14C-naphthalene, 14C-phenanthrene and 14C-pyrene mineralisation over 14 d in respirometric assays. The presence of other contaminants influenced the ability of the indigenous microflora to mineralise structurally different contaminants over time. 14C-Naphthalene mineralisation was inhibited by the presence of other contaminants; whereas the presence of naphthalene significantly enhanced rates of mineralisation in multiple-contaminant systems containing 14C-phenanthrene and 14C-pyrene. Generally, increasing the number of contaminant applications has implications for catabolic activity of soil microbes. It is suggested the toxic nature of PAHs retarded mineralisation at increased contaminant concentrations. The simultaneous effects of PAH concentration, contaminant mixture and repeated application on the development of catabolic activity in soil.