Background Men who have sex with men (MSM) are at risk of HIV and are an important population to monitor and ameliorate combination prevention efforts. Aim To estimate HIV prevalence and identify factors associated with frequent HIV testing (≥ 2 HIV tests in the last year) and pre-exposure prophylaxis (PrEP) use among MSM in London. Methods For this cross-sectional study, MSM recruited from 22 social venues provided oral-fluid samples for anonymous HIV antibody (Ab) testing and completed a questionnaire. Factors associated with frequent HIV testing and PrEP use were identified through logistic regression. Results Of 767 men recruited, 545 provided an eligible oral specimen. Among these, 38 MSM (7.0%) were anti-HIV positive including five (13.2%; 5/38) who reported their status as negative. Condomless anal sex within the previous 3 months was reported by 60.1% (412/685) men. Frequent HIV testing was associated with, in the past year, a reported sexually transmitted infection (adjusted odds ratio (AOR): 5.05; 95% confidence interval (CI): 2.66–9.58) or ≥ 2 casual condomless partners (AOR 2–4 partners: 3.65 (95% CI: 1.87–7.10); AOR 5–10 partners: 3.34 (95% CI: 1.32–8.49). Age ≥ 35 years was related to less frequent HIV testing (AOR 35–44 years: 0.34 (95% CI: 0.16–0.72); AOR ≥ 45 years: 0.29 (95% CI: 0.12–0.69). PrEP use in the past year was reported by 6.2% (46/744) of MSM and associated with ≥ 2 casual condomless sex partners (AOR: 2.86; 95% CI: 1.17–6.98) or chemsex (AOR: 2.31; 95% CI: 1.09–4.91). Conclusion This bio-behavioural study of MSM found high rates of behaviours associated with increased risk of HIV transmission. Combination prevention, including frequent HIV testing and use of PrEP, remains crucial in London.