The parameters that affect bicyclists’ satisfaction are of vital importance when it comes to determining the reasons that endure bicycle usage in an urban environment. This research refers to the factors that affect bicycle users’ satisfaction with the existing infrastructure in the Municipality of Thessaloniki, Greece. Bicycle dependence in Thessaloniki is relatively limited when it is compared to other European cities with similar topological and demographic characteristics. This article aims to determine the most suitable measures that policymakers should implement to enhance bicycle infrastructure. The data collection process was realized through an online questionnaire survey addressed to the bicycle organizations and groups that are situated in Thessaloniki. Inferential statistical analysis investigated the dependency between the perceived satisfaction and the perceived level of safety of cyclists. Ordinal and multinomial logistic regressions were applied to identify the significant problems that influenced cyclists’ satisfaction, as well as the most important improvement measures suggested by the survey participants. The findings revealed that the lack of safety and urban integration of bicycle infrastructures were the statistically significant issues affecting users’ satisfaction. The development of a safe, integrated, and interconnected bicycle network area emerges as a priority to increase cyclists’ satisfaction and daily bicycle trips in cities with low bicycle culture.