Background Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit upon the host. Knowledge and attitudes of health professionals have been reported to be at a medium level for probiotics . The objective was to evaluate the knowledge and practice styles about probiotics among pediatricians working in different regions of Saudi Arabia. Methods This cross-sectional study was conducted at pediatric hospitals in Saudi Arabia. A national survey of 550 pediatric providers (PPs) was conducted between January and March 2020 anonymously on their knowledge and practice styles regarding probiotics, and it was completed by pediatric residents (PRs), pediatric specialist (PSs), pediatric consultants (PCs), and pediatric gastroenterologists (PGs). Results The survey had a response rate of 82%. Among the respondents, 57.7% were aware of the probiotic’s definition. There were significant differences in the percentage of participants who had little knowledge of probiotics (P<0.05), with the highest being PRs and the lowest being PGs. The most common probiotic used by all participants was Lactobacillus acidophilus (63.3%), and Mycobacterium avium was prescribed the least often (8.6%). Most PRs and PSs correctly reported that probiotics reduce the risk of antibiotic-induced diarrhea (74.9% and 80.2%, respectively), but there were no significant differences among them. Conclusions Significant differences in knowledge and practice patterns exist for probiotics. Identification of knowledge gaps may be useful to develop educational materials to improve the proper definition, knowledge, and use of probiotics.