Background: In healthcare, research is essential for improving disease diagnosis and treatment, patient outcomes, and resource management, while fostering evidence-based practice. However, conducting research in this sector can be challenging, and healthcare workers may face various obstacles while engaging in research activities. Therefore, understanding healthcare workers’ attitudes toward research participation is essential for overcoming barriers and increasing research engagement. In this study, these aspects are examined through the analysis of survey data from a tertiary healthcare institution in Saudi Arabia. Method: Data obtained via a survey conducted between April and November 2022 among the healthcare workers and employees at a tertiary care hospital in Saudi Arabia were analyzed using descriptive and bivariate statistics. Results: The study sample comprised 713 respondents, 61.71% of whom were female, 58.06% were 26–41 years old, and 72.93% had not undertaken any research as employees or affiliates. A significant association was noted between age group and time constraints (p = 0.004) and lack of opportunity for research (p = 0.00), which were among the identified barriers to research participation. A significant association was also found between gender and barriers to pursuing research (p = 0.012). When the 193 (27.07%) participants who conducted research were asked about the challenges they encountered during this process, gender was significantly associated with difficulties in allocating time for conducting research (p = 0.042) and challenges in accessing journals and references (p = 0.016). Conclusion: The study findings highlight the importance of addressing the barriers and challenges in promoting positive attitudes toward research participation among healthcare workers considering their gender and age. In this manner, healthcare institutions can adopt an environment conducive for professional research engagement.