ABSTRACT Metarhizium rileyi is a biological agent used in the control of various pests, particularly noctuid insects. However, this fungal species is slow-growing, resulting in a prolonged culture period and limiting its commercial production. In this study, 0.3% (w/w) oleic acid significantly enhanced the rates of hyphal growth, conidial production, and conidial germination by 23%, 9%, and 10%, respectively. The expression of genes related to microbial growth and development (MrPbs2, MrMsn2, MrSwi6, MrNsdD, Mrap1 and MrSte12) was significantly increased in the presence of oleic acid. The M. rileyi conidial production and its effects on the mortality of Spodoptera frugiperda were investigated using rice-vegetable oil solid medium (RVOSM) and its substitute Sabouraud maltose agar plus yeast extract (SMAY) plates containing 0.03% oleic acid. The fungi retained their toxicity to S. frugiperda. Therefore, oleic acid can improve the growth potential of fungi, and RVOSM can be used as a substrate for conidia production for the application of M. rileyi as a biocontrol agent.