Entomopathogenic nematodes (EPNs) are crucial biocontrol agents, yet optimizing the yield and quality of infective juveniles (IJs) during commercial liquid production remains challenging. This study utilized a central composite rotatable design to optimize liquid culture parameters (ascaroside, dimethyl sulfoxide, medium volume, IJ inocula) for Heterorhabditis bacteriophora H06 and Steinernema carpocapsae All. The results demonstrated that improving aeration (inferred from reduced media volume), combined with ascr#11 regulation, synergistically enhanced IJ yield and quality. Under optimized conditions, yields reached 3.35 × 10⁵ IJs/mL for H. bacteriophora H06 and 2.67 × 10⁵ IJs/mL for S. carpocapsae All. Crucially, the IJs from the high-yield flask exhibited significantly superior infectivity (24–26% single-IJ infection rate) compared to solid-culture controls (13–14%). Targeted metabolomics profiling of sugar, energy and fatty acids of H. bacteriophora H06 revealed upregulated tricarboxylic acid (TCA) cycle intermediates (citrate, pyruvate) and the significant accumulation of stress-protectant trehalose and immune-modulating polyunsaturated fatty acids (eicosapentaenoic acid, arachidonic acid). These findings establish a fermentation strategy that simultaneously enhances IJ yield and biological quality by reducing media volume (used as a proxy for improved aeration) and supplementing ascr#11. Furthermore, the distinct metabolic profile enriched in energy, stress, and immune-modulating metabolites identified in H. bacteriophora provides a plausible explanatory framework for the parallel phenotypic improvements observed across both species.