Ecotourism is a type of tourism based on the principle of sustainability, aiming for social and ecological development while preserving natural and cultural values. Accurate information transfer and effective promotion play a key role in the success of this type of tourism. Tour guides have a critical role in ensuring the sustainable implementation of ecotourism and increasing tourist satisfaction at this point. The performance of tour guides directly affects the quality of tourists’ experiences, contributing to the destination’s image. This study examines how tourists evaluate the roles of tour guides working in the field of ecotourism. The research was conducted in April 2023 in the Muğla province (Bodrum, Marmaris, Muğla City Center, and Milas) with 132 ecotourists using a questionnaire. Tourists’ expectations from tour guides were analyzed in terms of demographic variables such as nationality, gender, age, and ecotourism experience. The findings have indicated that tour guides’ environmentally sensitive behaviors, support for local communities, and adherence to sustainable tourism principles increase tourist satisfaction from their ecotourism experience. The study offers recommendations for tour guides, travel agencies, and destination managers, aiming to raise awareness and improve the quality of guiding service in ecotourism.