Biochar, a low-cost carbonaceous product, is gaining relevance in the remediation of hydrocarbon-contaminated soil. Current literature has shown that biochar studies have been carried out under different conditions. Although some attempts have been made to assess the effect of varying conditions in biochar-based remdediation studies, no work has assessed the effect of biochar pyrolysis temperature, biochar dose, and fertiliser dose altogether on the efficacy of biosolids-derived biochar in the remediation of diesel-contaminated soil, despite the fact that the influence of these parameters on the efficacy of the remediation process are likely to be significant. This study aimed to investigate the effect of biosolids-derived biochar on the remediation and ecotoxicity of diesel-contaminated soil, as well as to assess the influence of biochar pyrolysis temperature, biochar dose, and fertiliser addition on soil remediation. After 12 weeks, the contaminated soil amended with biochar produced at 900 °C and applied at 10% together with 1% fertiliser had a TPH concentration lower than the EPA Victoria maximum threshold for Category D waste (5000 mg/kg); in contrast, the TPH concentration in the control exceeded this threshold. Further, soil ecotoxicity at week 12 was lower in most of the biochar treatments. The alkB gene copy numbers increased at week 12 in almost all treatments. Hydrocarbon removal and soil ecotoxicity was affected by the studied factors. This study demonstrated the potential of biosolids biochar as a low-cost treatment to enhance the bioremediation of diesel-contaminated soils, while showing the importance of the treatment conditions on the biochar efficacy in remediation.