Analyzing ambiguous bite marks using conventional morphological approaches to identify attackers is difficult; thus, applying molecular biological methods for identifying an attacker from their saliva is a possible approach in a forensic investigation. This study aimed to establish oral bacterial DNA-based human and canine saliva markers and develop a practical method for their discrimination. We considered Streptococcus oralis and Pasteurella canis as human and canine saliva marker candidates, respectively. Duplex bacterial DNA detection using melting curve analysis was designed and evaluated for forensic applicability using proof-of-concept experiments. S. oralis DNA was detected from human saliva samples from 30 out of 30 individuals, and P. canis DNA was detected from canine saliva samples from 73 out of 77 individuals (26 dog breeds). Additionally, both bacterial DNA markers were accurately detected from human blood-contaminated saliva samples and mock indistinct bite marks. Our results indicate that both bacterial DNA markers were sensitive, robust, and discriminating saliva markers. We consider that our duplex bacterial DNA examination is a simple, practical, and useful method for the detection of saliva from indistinct bite marks and discrimination between human and canine saliva.