Technical Abstract: The use of the selective inhibition method for measuring fungal-to-bacterial ratios may be limited due to biocide selectivity and the overlap of antibiotic activity. This study was conducted to evaluate novel pairs of antibiotics for their specificity in soils of different origins and their potential reduction in inhibition of non-target organisms. Four soil selected for this study were from a semi-arid shrub-steppe, a loblolly pine forest soil, and two grassland soils (restored and farmed prairie soils). Three bactericides were tested: oxytetracycline hydrochloride, streptomycin sulphate, and bronopol. Three fungicides were tested: captan, ketoconazole, and nystatin. The inhibitor additivity ratio and fungal to bacterial ratios were calculated from control and treated soils where inhibition was measured as CO2 respiration reduction with biocides. We were best able to minimize non-target inhibition by the antibiotics to less than 5% and thus calculate reliable fungal-to-bacterial ratios using captan to inhibit fungi in all four soils, and bronopol to inhibit bacteria in three of the four soils. The most successful bactericide in the restored prairie was oxytetracycline-HCl. Our paper demonstrates that application of novel antibiotics is not uniformly successful in soils of different origin and that the SIR selective inhibition technique requires more than just optimization of antibiotic concentration; it also requires optimization of antibiotic selection.