Purpose <p>The primary challenge in bioremediating weathered diesel oil (WDO) lies in the initial biological lag phase and the low bioavailability of hydrocarbons. While exogenous surfactants are conventionally applied to rapidly solubilize WDO into the aqueous phase, this approach is fundamentally limited by severe surfactant-induced microbial toxicity. This study engineered a novel biostimulation formulation that seamlessly integrates initial exogenous solubilization with subsequent endogenous biosurfactant production. A mixed-micellar system was developed to provide immediate chemical solubilization while effectively buffering the inherent toxicity of exogenous surfactants. The formulation strategically incorporates Mg(II) to specifically stimulate indigenous microbes to secrete endogenous biosurfactants following the lag phase. This dual-action design aims to overcome toxicity barriers, accelerate microbial proliferation, and achieve efficient WDO degradation.</p> Materials and methods <p>A novel micellar shielding system was engineered by specifically combining sodium dodecyl sulfate (SDS) and Tween 80 to physically sequester anionic toxicity while maintaining robust solubilization capacity. Batch solubilization and biocompatibility experiments were conducted to evaluate this structural advantage on microbial viability and total petroleum hydrocarbon (TPH) removal. Mg(II) and cost-effective carbon sources were integrated to specifically trigger in situ endogenous biosurfactant production. A Taguchi experimental design was employed to optimize these formulation parameters. Remediation mechanisms were investigated by correlating TPH removal dynamics with functional gene (<i>alkB</i>) expression and shifts in microbial population structure. The optimized synergistic formulation was validated in a mesocosm-scale biopile system.</p> Results and discussion <p>Biocompatibility tests showed that SDS alone exhibited significant microbial toxicity, whereas the addition of Tween 80 mitigated inhibition through mixed-micelle formation and provided a co-substrate effect. The optimal surfactant-nutrient synergy using <i>Pseudomonas aeruginosa</i> Tar3 as a model organism for was developed for mechanistic optimization. Batch experiments demonstrated that 1–2% Tween 80 combined with 0.25% SDS enhanced TPH removal to approximately 55–60%, outperforming SDS alone (&lt; 40%). The optimized formulation (1% Tween 80, 0.25% SDS, 6× Mg(II), and 1.5% molasses) achieved a TPH removal efficiency of 72.4% in the biopile system over 112 days, accompanied by 2 to 3 orders of magnitude increases in total bacterial counts and <i>alkB</i> gene abundance.</p> Conclusions <p>This study successfully engineered a surfactant formulation that overcomes challenges of hydrocarbon bioavailability and microbial toxicity in the bioremediation of WDO-contaminated soils. The synergistic combination of anionic and nonionic surfactants through mixed-micelle formation mitigated SDS-induced cellular toxicity while maintaining high solubilization efficiency. This approach provides a scientifically validated, cost-effective, and environmentally-compatible strategy for the remediation of recalcitrant petroleum hydrocarbon–contaminated soils.</p>