Biological control agents are a promising substitute for chemical pesticides in agricultural pest management. In this study, Penicillium sp. with high pathogenicity to the agricultural pests oriental leafworm moth (Spodoptera litura) and diamondback moth (Plutella xylostella) were isolated from naturally infected insects and grown on different agricultural residues as an inexpensive substrate for their sporulation. Ten strains of Penicillium (P.01~P.10) were identified as P. citrinum based on morphological features and molecular studies, with sequence analysis using an internal transcribed spacer region. Different fungal isolates exhibited a varying degree of pathogenicity against S. litura and Pl. xylostella, and strains P.04 and P.09 showed the highest pathogenicity to S. litura, with a mortality rate of 92.13% after 7 days of treatments, while strain P.06 resulted in the highest mortality of Pl. xylostella (100%) after 6 days of treatment. Moreover, among ten isolates infected with both S. litura and P. xylostella, P.06 showed potential virulence against S. litura and Pl. xylostella, with lethal time for 50% mortality (LT50) values of 4.5 days and 3.0 days, respectively. The ten isolates showed higher virulence to Pl. xylostella than to S. litura. The agro-industrial-based medium showed efficiency for the cultivation of isolates for sporulation on an industrial scale, suggesting that the newly isolated P. citrinum is a potential biological control agent for controlling insect pests and could be further developed for microbial pesticide production.