We assessed sexual risk behaviours, condom use and sexually transmitted infection (STI) treatment-seeking behaviours among truckers and female sex workers (FSWs) operating at 12 hotspots along two major transport corridors in Uganda. Participants were identified through local contact persons: 261 truckers and 259 FSWs were interviewed. FSWs reported a mean number of 28 partners in the past 30 days while truckers reported a mean of seven partners. Ninety-four percent of FSWs and 87% of truckers reported condom use in the past month; however, only 21% of truckers and 45% of FSWs reported using condoms consistently during that time. More than half of truckers (n = 261) and 77% of FSWs (n = 259) reported that they suffered from STIs in the past year, and 93% of FSWs and 92.9% of truckers sought treatment for STIs in the past year. Of these, 78% of truckers and 86% of FSWs sought treatment at least three days following recognition of symptoms. These findings suggest that FSWs and truckers engage in high-risk sexual behaviours characterized by multiple sexual partnerships, low consistent condom use and delayed treatment-seeking behaviours.