Aim: To measure and analyze knowledge and attitudes among selected groups of health professionals. Methods: The research was conducted from March 3rd to August 5th. 2024. using the survey questionnaire "Knowledge and attitudes about probiotics", which consisted of three parts. The first part evaluated the sociodemographic data of the participants, the second part assessed their knowledge and the last part addressed the attitudes of participants toward probiotics (18 statements). The sample included 475 participants (a total of 352 Croatian and 123 Slovenian respondents) who had received education in the field of biomedicine and healthcare. Results: The average age of respondents was 35,6 ± 10,8 years, of which 76% were women and 24% were men. The largest group of health professionals was nurses (41,1%), followed by physicians (17,9%) and pharmacists/pharmaceutical technicians (15,4%). The majority (61,1%) demonstrated sufficient level of knowledge about probiotics, whilst almost a third of the respondents (32,4%) had an insufficient level of knowledge and only 6,5% of them exhibited good level of knowledge. Women and the more educated had statistically significant higher level of knowledge and more positive attitudes. Respondents with longer working experience achieved greater knowledge, while there was no statistically significant difference in attitudes among them. There was a significant difference in knowledge between different professions (p<0,05). Respondents with the pharmaceutical background showed a statistically significant higher level of knowledge and more positive attitudes compared to other respondents with different professions. Slovenian respondents had a statistically significant higher level of knowledge and more positive attitudes. Most respondents (77,9%) identified the correct definition of probiotics. The most frequently recognized organisms with probiotic strains were Lactobacillus acidophilus (93,7%) and Bifidobacterium bifidum (71,2%). More than half of respondents had a positive attitude (69,5%). Furthermore, those with a higher level of knowledge about probiotics had statistically significant more positive attitudes. 87,2% of respondents didn't fear about introducing probiotics into their body. Only 16,8% of respondents believed that healthcare professionals are sufficiently educated to advise on the use of probiotics, whilst 3,8% of them thought that only doctors (physicians) should learn about probiotics, yet 78,3% of respondents expressed a desire to learn more about probiotics. Conclusion: Despite the positive attitude of most health professional respondents (69,5%), only 6,5% of them demonstrated a good level of knowledge about probiotics. The result suggests further scope for implementation of targeted educational programs of healthcare professionals about probiotics, particularly taking into account the fact that they hold the guidelines and establish best evidence-based practice standards in health related field.