Objective Female sex workers (FSWs) are vulnerable to human alphaherpesvirus 2 (HSV-2) infection due to their high numbers of sexual partners. The objective of this study was to evaluate the seroprevalence and risk behaviours associated with HSV-2. Methods A cross-sectional study was conducted in Mato Grosso do Sul, Brazil. A total of 376 FSWs were recruited by respondent-driven sampling (RDS) methodology and answered an epidemiological questionnaire. Blood samples were collected to test for HSV-2 antibodies using commercial ELISA and for HSV-2 DNA using real-time PCR. Results The seropositivity was 47.3% (178/376) for HSV-2 IgG and 10.1% (38/376) for HSV-2 IgM. HSV-2 viraemia was detected in two infected FSWs with primary infections. In bivariate and multivariate analyses, the OR for HSV-2 IgG increased with age (OR=2.53–7.90, OR=2.66–6.37) and the number of sexual partners (OR=2.30–3.25). On the other hand, daily alcohol consumption (OR=0.10) and the use of condoms during the last intercourse (OR=0.47) were protective factors against HSV-2 acquisition. Conclusion Despite the impact of FSWs in public health policies with the dissemination of sexually transmitted infections, there have been few studies performed regarding the prevalence of HSV-2 in Brazil, making it difficult to implement any control or preventative measures. The results produced here using an RDS methodology demonstrated a high prevalence, risk behaviours and primary infection among the FSWs. These results reinforce the need to implement control and preventative measures for HSV-2 infection in this population.