The striped flea beetle (SFB), Phyllotreta striolata, is a major pest of Brassicaceae crops, causing substantial yield losses worldwide. Effective biocontrol strategies, particularly the development of mycoinsecticides, require the identification of virulent entomopathogenic fungi (EPF) and the establishment of reliable bioassay systems. However, establishing reliable bioassay systems for SFB has been particularly challenging, especially for larval stages due to their recalcitrant rearing requirements. This study aimed to establish a standardized bioassay protocol to evaluate EPF efficacy against SFB. A specialized larval collection apparatus was developed, and the virulence of three EPF strains (Beauveria bassiana BbPs01, Metarhizium robertii MrCb01, and Cordyceps javanica IjH6102) was assessed against both adult and larval stages using a radish slice-based rearing system. Intriguingly, BbPs01 and MrCb01 exhibited significantly higher LT50 values in larvae than in adults, contrary to the typical pattern of greater larval susceptibility observed in most insect systems. We hypothesized that isothiocyanate—specifically sulforaphane, a compound abundant in radish tissues—exerts fungistatic effects that impair fungal growth and virulence. Follow-up experiments confirmed that radish-derived sulforaphane inhibited fungal activity. Through alternative host plant screening, Chinese flowering cabbage (Brassica campestris L. ssp. chinensis var. utilis) was identified as an optimal larval diet that minimally interferes with EPF bioactivity, enabling reliable virulence assessments. This study presents critical methodological advancements for SFB biocontrol research, providing a robust framework for standardized larval bioassay and novel insights into plant secondary metabolite interactions with entomopathogens. The optimized system supports the development of targeted mycoinsecticides and contributes to a deeper understanding of tri-trophic interactions in crucifer pest management.