The increasing demand for efficient juice extraction processes in response to rising global fruit product consumption underscores the importance of enzyme-mediated approaches, particularly utilizing pectinases, in enhancing extraction efficiency by breaking down plant cell wall components. This study focuses on investigating pectinase production from different Aspergillus spp. strains isolated from natural resources in Ankara, Turkey. By screening these strains, this research aims to broaden the spectrum of working conditions adaptable to diverse extraction processes. Emphasizing optimal performance in acidic environments and high temperatures prevalent in fruit juice extraction, the study isolates, and screens Aspergillus spp. strains, conducts pectinase assays, and identifies the most promising strain, Aspergillus brasiliensis, with substantial enzyme activity. Fermentation experiments further elucidate optimal conditions for enzyme production. Results reveal significant enzymatic activity under acidic and high-temperature conditions, with the chosen isolate exhibiting remarkable enzyme activity and specific activity at varying temperatures and nitrogen sources. This investigation highlights the potential of different strains and their enzymatic capabilities to propel innovation in various biotechnological domains, promising advancements in fruit processing and related industries.