Waste separation is a crucial step toward sustainable waste management. Understanding public perception and participation in this process is essential for its success. This study evaluates public perception and participation in the selective collection of general waste and biowaste in Guarda, Portugal, identifying key motivations, barriers, and areas for improvement. A cross-sectional analytical study was conducted using an online survey with 205 residents. Results indicate that 83.4% (95% CI: 77.9%-88.0%) of participants separate waste in the household, while workplace separation is lower (65.9%, 95% CI: 59.2%-72.1%). Paper and cardboard are the most frequently separated materials, whereas used cooking oil receives the least attention (95% CI: 16.5%-30.6%). Regarding biowaste, 92.2% (95% CI: 87.9%-95.3%) of respondents understand the concept, and all recognize its environmental benefits. However, 43.4% (95% CI: 36.8%-50.3%) are unaware of the municipal biowaste collection network, and 57.1% (95% CI: 50.2%-63.7%) find the existing infrastructure inadequate. Municipalities should expand and optimize waste collection systems to improve participation while strengthening public awareness campaigns. Social media is the preferred information channel (91.2%, 95% CI: 86.8%-94.5%), but traditional outreach methods, such as school initiatives and community meetings, could enhance engagement. Addressing infrastructure gaps and promoting consistent separation practices across different settings are key to optimizing waste management. This study highlights the need for improved public engagement and infrastructure to ensure the success of selective waste collection, particularly for biowaste, contributing to enhanced sustainability efforts in the region.