OBJECTIVES Disc diffusion is still largely based on manual procedures. Technical variations originate from inoculum preparation, variations in materials, individual operator plate streaking and reading accuracy. Resulting measurement imprecision contributes to categorization errors. Biological variation resembles the natural fluctuation of a measured parameter such as antibiotic susceptibility around a mean value. It is deemed to originate from factors such as genetic background or metabolic state. This study analysed the relative contribution of different technical and biological factors to total disc diffusion variation. METHODS For calculation of relative error factor contribution to disc diffusion variability, five experiments were designed keeping different combinations of error factors constant. A mathematical model was developed to analyse the individual error factor contribution to disc diffusion variation for each of the tested drug-species combinations. RESULTS The contribution of biological variation to total diameter variance ranged from 10.4% to 98.8% for different drug-species combinations. Highest biological variation was found for Enterococcus faecalis WT and vancomycin (98.8%) and for penicillinase-producing Staphylococcus aureus and penicillin G (96.0%). Average imprecision of automated zone reading revealed that 1.4%-5.3% of total imprecision was due to technical variation, while materials, i.e. antibiotic discs and agar plates, contributed between 2.6% and 3.9%. Inoculum preparation and manual plate streaking contributed 6.8%-24.8% and 6.6%-24.3%, respectively, to total imprecision. CONCLUSIONS This study illustrates the relative contributions of technical factors that account for a significant part of total variance in disc diffusion. The highest relative contribution originated from the operator, i.e. manual inoculum preparation and plate streaking. Further standardization of inoculum preparation and plate streaking by automation could potentially increase the precision of disc diffusion and improve the correlation of susceptibility reports with clinical outcome.