The objective of this study was to survey health professionals to investigate their knowledge of probiotics. An online survey was conducted to gather data on the knowledge of health professionals. The online survey was distributed via email and social media platforms using snowball sampling. A total of 1066 health professionals (859; 80.6% female) from 30 countries responded to the survey. Most of the respondents evaluated their knowledge of probiotics as medium (36.4%) or good (36.2%). Only 8.9% of the respondents rated it as excellent. No statistical difference in knowledge was found between male and female health professionals. Over 80% of pharmacists, allied health professionals, medical doctors and dentists, and other health professionals knew the correct definition of probiotics as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host", whereas three quarters of registered nurses and midwives and less than two thirds of psychologists identified the correct definition. Statistically, more female than male health professionals knew the correct definition of probiotics. The most frequently recognized species of bacteria containing probiotic strains were (92%), (82%), and (62%). The opinions on when it is best to take probiotics were different (χ = 28.375; p < 0.001), with 90.2% of respondents identifying that probiotics have beneficial effects if taken during antibiotic therapy, 83.5% for diarrhea, 70.6% for constipation, 63.3% before traveling abroad, and 60.4% for treating allergies. Almost 79% of health professionals involved in this study have advised their patients to use probiotics and 57.5% of the respondents wanted to learn more about probiotics. All things considered, health professionals have a medium level of knowledge of probiotics, which could be improved by the implementation of targeted learning programs. As probiotics have many beneficial effects in a wide range of health areas, health professionals need to adopt the use of probiotics in clinical practice.