Insectivorous bats may significantly contribute to human well-being by suppressing pest insects and possibly preventing the emergence of diseases. To understand the roles these bats play in their ecosystems, a diet analysis of their guano can be carried out. However, each diet analysis method has its drawbacks, e.g., some soft-bodied insects might be missed in microscopic analysis of guano. We aimed to examine the diet of the wrinkle-lipped free-tailed bats (Chaerephon plicatus) using direct PCR and DGGE (denaturing gradient gel electrophoresis) technique. Sequencing was done for 240 guano samples collected from two caves in Thailand over the course of a year. Direct PCR and DGGE was successfully applied for bat guano analysis. Seventy-six Operational Taxonomic Units were identified, in which 25 were determined to the species level. Diptera was the most abundant insect order found in bats' diet, with a percentage frequency of occurrence (%FOO) of 32.8%, followed by Hemiptera (27.2%), Lepidoptera (24.1%) and Coleoptera (10.3%). Hemipterans were preferred during active rice-growing months, while dipterans were consumed year-round. Eight known crop pests were found, and the brown planthopper (Nilaparvata lugens) was the most dominant throughout the year. Mosquitoes also substantially contributed to the bat's diet. The bats probably encounter these insects during insect dispersal at high elevations. The prey species recorded strongly indicated that this bat plays a role in facilitating rice and crop productions, which increases food security. In addition, this bat may play a role in suppressing potential disease-carrying insects such as various species of mosquitoes. We urge local and international authorities to increase conservation efforts and that similar studies should be done with other bat species.