It could be a rival cowboy named Orochimaru. He's jealous of Sasuke's skills and the fact that Sakura is with him. Orochimaru might try to steal Sasuke's horse or sabotage their quests for supplies. He could also spread false rumors about them in the small Western towns they pass through, causing trouble for them.
Maybe a group of outlaws led by Kabuto. They see Sasuke and Sakura as easy targets. They try to rob them of their valuables, which include a special lemon - scented medicine that Sakura has made. This medicine is important for a sick child in a nearby town. So, Sasuke and Sakura have to fight off these outlaws to protect the medicine and continue their journey.
The main antagonist could be a land - greedy rancher named Danzo. He wants to take over all the land in the area, including the land where the lemon trees grow that Sakura loves. He tries to drive Sasuke and Sakura out of the region by any means necessary, setting up traps for them and hiring thugs to attack them.