Development of rapid and multiplexed diagnostic tools is a top priority to address the current epidemic problem of sexually transmitted diseases. Here we introduce a novel nanoplasmonic biosensor for simultaneous detection of the two most common bacterial infections: Chlamydia trachomatis and Neisseria gonorrhoeae. Our plasmonic microarray is composed of gold nanohole sensor arrays that exhibit the extraordinary optical transmission (EOT), providing highly sensitive analysis in a label-free configuration. The integration in a microfluidic system and the precise immobilization of specific antibodies on the individual sensor arrays allow for selective detection and quantification of the bacteria in real-time. We achieved outstanding sensitivities for direct immunoassay of urine samples, with a limit of detection of 300 colony forming units (CFU)/mL for C. trachomatis and 1500 CFU/mL for N. gonorrhoeae. The multiplexing capability of our biosensor was demonstrated by analyzing different urine samples spiked with either C trachomatis or N. gonorrhoeae, and also containing both bacteria. We could successfully detect, identify and quantify the levels of the two bacteria in a one-step assay, without the need for DNA extraction or amplification techniques. This work opens up new possibilities for the implementation of point-of-care biosensors that enable fast, simple and efficient diagnosis of sexually transmitted infections.