Cycling’s integration into the intricate facets of urban design, together with walking and public transportation, offers an effective solution to the mobility issues plaguing urban spaces, and is critical to the sustainability of modern cities. In this context, in this research urban cyclists’ needs and preferences are analyzed through questionnaires, and bicycle accessibility to urban areas is assessed using multicriteria analysis. The public’s familiarity with the integration of novel mobility solutions (e.g., e-bicycles) that support accessibility and inclusiveness is tested and analyzed by recording cyclists trajectories on bicycle routes. The European hub of Patras was selected as the case study for a pilot scheme in this analysis. Similar to many medium-sized European cities, several mobility obstacles, including urban topography, hinder bicycle accessibility in the city, especially between the Modern and Old City areas. The research findings indicate that, addressing these obstacles, electric bicycles can substantially increase bicycle accessibility in the city center. The public usage of electric bicycles is encouraged in the pilot study, and the results indicate that it can increase accessibility to urban areas while reducing restrictions related to age, physical condition, and disabilities. Providing citizens access to e-bicycles can increase the number of daily bicycle users, leading to positive impact in urban cohesion, resilience, and sustainability.