Background: There is limited documentation on knowledge, attitudes and barriers to condom use among female sex workers (FSWs) and truck drivers (truckers). Objective: To explore knowledge, attitudes and barriers to condom use among FSWs and truckers operating along major transport corridors in Uganda Methods: Structured questionnaires were administered to explore FSWs' and truckers' knowledge of and attitudes towards condom use among 259 FSWs and 261 truckers. Qualitative data were collected on barriers to condom use using focus group discussions. Quantitative data were analyzed using SPSS while qualitative data were audio-recorded, transcribed and thematically analyzed. Results: Condom knowledge was high with 97% of FSWs and 95% of truckers agreeing with the statement, "using condoms properly and consistently reduces risk of HIV infection". Attitudes towards condom use were generally favorable with 91% of FSWs and 82% of truckers agreeing with the statement, "condom use is the best method of HIV prevention". Qualitative findings show that poverty, refusal to use condoms by male partners, alcohol use before sex and beliefs that condoms 'kill the mood for sex' remain key barriers to consistent condom use. Conclusions: Consistent condom use among FSWs and truckers is still hampered by economic and relationship factors.