Background: In scientific literature, it is relatively rare to find information on healthcare professionals’ current knowledge of probiotics, prebiotics, and gut microbiota. Objective: The aim of our study was to assess healthcare professionals’ knowledge of gut microbiota, probiotics, and prebiotics in the city of Kénitra, Morocco. Material and Methods: The data was collected via an online questionnaire, which we distributed through social media. A total of 143 healthcare professionals (78.3% women and 21.7% men) responded to this questionnaire. The questionnaire concerned knowledge of probiotics and prebiotics. Results: Most respondents rated their knowledge of probiotics, prebiotics, and gut microbiota as average (40%) or poor (39%), while others rated their knowledge as good (11%) and only 2% had very good knowledge, with the remainder (8%) having no knowledge. The correct definition of probiotics chose 67.1% of respondents, broken down as follows: 80.4% of general practitioners, 76.9% of specialists, and 57% of nurses. Lactobacillus acidophilus (65%) and Bifidobacterium bifidum (50.3%) are the two species best known to respondents as probiotic strains. Furthermore, the most popular prebiotic is fructooligosaccharide (51%), followed by galactooligosaccharide (42.7%) then inulin (36.4%) and finally betaglucan (14%). Among professionals 60.1% prescribed probiotics and/or prebiotics for diarrhea, followed by antibiotics (47.6%) then constipation (39.2%) and 21% of respondents recommended them for diabetes, 18.9% for obesity while only 3.5% used them for other pathologies. Conclusions: This online survey revealed the current knowledge of healthcare professionals regarding probiotics, prebiotics and gut microbiota and highlights the importance of educating and training them through targeted learning programs.