The biocontrol potential strain of Penicillium ochrochloron Q-3-1 was isolated from Hepialus armoricanus larvae. However, the insecticidal and antifungal mechanisms of Q-3-1 are still unclear. Eleven chitinase genes (Poch1-Poch11) were isolated for functional identification. During Q-3-1 infection, Poch1, Poch2, and Poch11 were upregulated in Galleria mellonella, whereas Poch4, Poch6, and Poch11 were upregulated in Diaphorina citri. Chitinase activity was determined based on the expression of the chitinase genes in an in vitro prokaryotic expression system and an analysis of the p-nitrophenol (pNP) degradation ability of recombinant chitinase. They exhibit excellent degradation activities for chitin, citrus pectin, D-cellobiose, and G. mellonella epidermis. The exonuclease activity of Poch1 can effectively degrade insect chitin and pectin. The insect chitin utilization rate of Poch11 is higher than that of pectin, and it can randomly degrade different pNP substrates. In addition, the present study found that exogenous chitinase proteins showed significant antagonistic ability against citrus pathogens. The special antifungal and insecticidal character of Q-3-1 may lead to its potential application in agriculture as a biological control agent.