This study assessed food safety knowledge and practices (KP) among 461 university students in Jordan, comparing health-related and non-health-related students. A cross-sectional survey using a questionnaire evaluated demographics, food safety knowledge, and handling practices. Health-related students scored higher in food safety knowledge with significant differences between them (mean: 4.95 ± 2.32) compared to non-health-related students (mean: 3.73 ± 1.92, p < 0.001). Health-related female students also demonstrated better knowledge (mean: 6.70 ± 1.95) than males (mean: 6.03 ± 2.38, p = 0.008). Significant differences were found, especially among non-health-related students. Common issues included inadequate food storage knowledge and reliance on feel-touch tests instead of thermometers, leading to undercooking and foodborne infections. Additionally, the COVID-19 pandemic temporarily improved practices, though post-pandemic declines were noted in handwashing and thermometer use. This study highlights the need for targeted interventions, particularly for male students not enrolled in health-related fields. Targeted educational programs and practical training focusing on food safety can significantly improve both knowledge and practice, reducing health risks. Including food safety modules in non-health disciplines can help bridge the gap between academic knowledge and practical application.