For entrepreneurial organizations to be able to successfully develop the business strategy, it is necessary to provide optimal resources for unhindered growth, internal coherence, stability of organizational culture, and competitiveness in the market. At the heart of these processes are employees, who directly influence organizational outcomes with their daily efforts, ability, and thinking. Also, the improvement of business functions is necessary for the organization to be competitive with other organizations in the market. In this context, the effectiveness of knowledge management is one of the key aspects in ensuring the development of both individual and group performance. In today’s time of great challenges for entrepreneurs, managing the processes that provide knowledge to both employees and organizations can be a key aspect of business sustainability that is imperative in turbulent circumstances. The purpose of the study was to examine how organizational culture and gender aspects of employees should be put in the function of knowledge management system (KMS). In line with this, this paper attempts to examine the extent to which the processes of improving the knowledge of employees from a gender perspective in entrepreneurial organizations in Serbia are effectively managed. In order for women to realize a greater contribution in entrepreneurial organizations, it is imperative to stimulate the educational process by empowering both women and men to improve their well-being and participate actively in entrepreneurial organizations building. Authors pointed out that when technology and organizational culture are coupled with educational assistance and training, however, adoption improves, and benefits are greater. Finally, they concluded that statistical parameters explicitly show that there are no significant differences on any tested issue, between males and females. This means that their perspective is identical when it comes to the development and use of knowledge management in entrepreneurial organizations.