Background <p>Omega-3 polyunsaturated fatty acids are essential nutrients with interventional benefits for cardiovascular, dermatological, and immune systems. Unexpectedly, few studies have investigated the implications of pharmacy knowledge and practice on omega-3 dispensing in pharmacies in Jordan.</p> Objective <p>This study aimed to explore the knowledge, attitudes, and practices of pharmacy students and pharmacists in Jordan concerning the health benefits of omega-3 fatty acids, patient counseling, supplement use, and prescription of omega-3 in Jordanian pharmacies.</p> Methods <p>A cross-sectional study utilizing a structured questionnaire was conducted between March and May 2025. The research team developed the study questionnaire, and independent researchers evaluated its face and content validity. To evaluate the questionnaire’s internal consistency, a pilot test was carried out. Three sections covering different topics of interest made up the final questionnaire. The questionnaire assessed demographic information, knowledge of omega-3 sources and health benefits, awareness of recommended dosages, and professional practices in supplement recommendation. Data were analyzed using SPSS.</p> Results <p>Omega-3 supplements are used by 60.5% (<i>n</i> = 477) of the participants and 65.4% (<i>n</i> = 516) by the participants’ family members. The mean knowledge score of omega-3 among the participants was 2.95 (SD = 5.28), while the Self-Assessment of Omega-3 Knowledge for the participants (45.4%) (<i>n</i> = 358) was low. The majority of respondents (74.1%) reported that omega-3 cannot be synthesized endogenously. While only 12.0% identified fish and plants as sources of omega-3. Likewise, only 16.3% of the participants identified the recommended dose of omega-3 (1000&#xa0;mg/day). Compared to students, pharmacists had higher percentages of correct responses for reducing the risk of preterm birth, incidence of depression, improving skin condition, and improving hair condition (<i>p-</i>value &lt; 0.05). Additionally, there was a significant increase in correct identification of both the recommended daily dose of omega-3 (<i>p-</i>value = 0.038) and the enhancement of hair condition (<i>p-</i>value = 0.008) among intermediate and expert pharmacists. Meanwhile, in multiple linear regression, gender was identified as the sole statistically significant predictor of the omega-3 benefits knowledge score (<i>p</i>-value = 0.014).</p> Conclusion <p>Pharmacists and pharmacy students in Jordan demonstrate good awareness of omega-3 sources and general benefits but exhibit notable gaps in knowledge of dosage, non-cardiovascular effects, and evidence-based recommendations. Educational interventions and professional training, especially for pharmacy students during the practical courses for undergraduates, are recommended.</p>