Are you trying to route out going traffic, or incoming traffic?
For Windows itself you can setup static routing rules (ROUTE ADD), however these won't apply to specific ports, and it only applies to outgoing traffic (incoming might use another route if it has already been discovered by outside equipment).
This really sounds like something you'll need to do on a switch (a managed switch, that is one that you can actually login into and program), or possibly on some models of router that support advanced traffic configuration.
One work around you might consider, if you are trying to connect to this computer from the outside (rather then run a connection from it) and this is a home setup, is to configure the port forwarding on your router to send traffic on that port to the IP address of the second card (you'll need to manually setup the IP of that card instead of using DHCP).
Alternatively a Linux computer running as a gateway could make it possible, but that would really just be a very expensive managed switch.