Probiotics are living microorganisms that, when consumed in sufficient quantities, provide health benefits to the individual. The level of knowledge and attitudes of community pharmacists toward probiotics has not been reported yet. Therefore, the aim of this study was to assess the knowledge and attitudes regarding probiotics among community pharmacists practicing in Aseer Region of Saudi Arabia. The study design involved a cross-sectional approach, where a questionnaire was used as the data collection tool. Descriptive analysis was conducted to analyze the sociodemographic data. Categorical variables were reported as frequencies and percentages, while continuous variables were presented as means and standard deviations. Out of 252 surveys distributed, 224 participants completed the survey (response rate: 88.88%). Most of the respondents were male (89.73%) who aged between 20 and 30 years (69.64%). More than half of the participants hold Pharm.D degree (53.13%) and most of them work in chain pharmacies (84.38%). The main source of participants information regarding probiotics was trusted medical sources (58.48%), followed by social media platforms (25.45%). More than half of the participants answered the definition of probiotics correctly (54.47%), and most of them know the difference between which bacterial strain may be harmful (75%). It is important to utilize the data for enhancing curricula in health programs and promoting the integration of clinical training on the use of probiotics alongside medical therapy.