Small and medium-sized enterprises are the backbone of the national economy, employment, and competitiveness, and contribute largely to the gross domestic product. The accession of the Slovak Republic to the European Union has created space for even more intensive cross-border cooperation with neighbouring countries. The most common areas of cross-border cooperation include strengthening of the economic, political, and cultural contacts. The aim of the paper was to identify the largest barriers to cross-border cooperation between companies of different size category and operating in different industry in the Prešov Region in the context of Slovakia - Poland cross-border cooperation. Information was obtained through a questionnaire survey and formulated hypotheses concerning barriers to Slovak-Polish cooperation were tested using selected mathematical-statistical methods. The results of the analysis show that businesses consider different currency to be the main barrier to cross-border cooperation, while the legislation related to doing business is perceived as the least problematic. The testing of the hypotheses shows that there is no statistically significant relationship between the size of the company and the perception of barriers in business development.