Example methods and systems for a computer system to perform security threat detection during service query handling are described. In one example, a process running on a virtualized computing instance supported by the computer system may generate and send a first service query specifying a query input according to a service protocol. The first service query may be detected by a security agent configured to operate in a secure enclave that is isolated from the process. Next, the security agent may generate and send a second service query specifying the query input in the first service query. It is then determined whether there is a potential security threat based on a comparison between (a) a first reply received responsive to the first service query and (b) a second reply received responsive to the second service query.