Introduction: There are few data on the acceptability of vaccination or blood sampling during Ramadan fasting month in Muslim countries. This could impact vaccination campaigns, clinical trials or healthcare during Ramadan. Methods: Using a semi-structured questionnaire, we conducted a cross-sectional study on 201 practising Muslims and 10 religious leaders in Conakry, Guinea in the wake of the recent epidemic Ebola epidemic. Acceptability of vaccination and blood sampling during Ramadan were investigated as well as reasons for refusal. Results: Vaccination was judged acceptable during Ramadan by 46% (93/201, 95% CI 0.40–0.53) of practising Muslims versus 80% (8/10, 95% CI 0.49–0.94) of religious leaders (p = 0.11). Blood sampling was judged acceptable during Ramadan by 54% (108/201, 95% CI 0.47–0.60) of practising Muslims versus 80% (8/10, 95% CI 0.49–0.94) of religious leaders (p = 0.19). The percentage of participants that judged both blood sampling and vaccination acceptable during Ramadan was 40% (81/201, 95% CI 0.34–0.47) for practising Muslims versus 80% (8/10, 95% CI 0.49–0.94) for religious leaders (p = 0.048). The most common reasons for refusal of vaccination or blood sampling were that nothing should enter or leave the body during Ramadan (43%), that adverse events could lead to breaking the fast (32%), that blood should not be seen during Ramadan (9%) and that the Quran explicitly forbids it (9%). Discussion: Although most Muslims leaders and scientists consider that injections including immunization and blood sampling should be authorized during Ramadan, many Muslims in our study judged vaccination or blood sampling unacceptable when fasting. Widely available recommendations on healthcare during Ramadan would be useful to inform Muslims.