Female sex workers (FSW) are considered one of the key affected populations in Papua New Guinea at risk of acquiring HIV. An integrated bio-behavioral survey of sex workers in Port Moresby was conducted to determine the nature and extent of this risk. About half (51.1 %) of the 411 FSW who reported having any sexual intercourse with clients had engaged in both anal and vaginal intercourse with clients in the last 6 months. In spite of having poorer HIV knowledge (OR95 % CI = 0.14–0.34), FSW who had anal intercourse with clients were significantly more likely to have used a condom at the last vaginal intercourse with a client (OR95 % CI = 1.04–2.87). Similarly, FSW who had anal intercourse with regular and casual partners were significantly more likely to have used a condom at the last vaginal intercourse. Those who engaged in both anal and vaginal intercourse with clients had similar condom use for both vaginal and anal intercourse, with the majority (78.1 %) using a condom at the last occasion for both vaginal and anal intercourse. These FSW may have different risk and protective factors that affect their use of condom during sexual intercourse. Further research is needed to investigate this difference between those who practice anal intercourse and those who do not in order to provide evidence for better programming.